bluuebunny/crossref_metadata_2025 huggingface dataset:
BINDING OF 37-DKa PLATELET AGGLUTINATING PROTEIN TO HUMAN PLATELETS
Authors: Lian E C -Y , Siddigui F A
Published: August 2018

We have previously reported the purification of a 37-KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura. using 125I-labeled p37, the properties of its binding to platelets were studied. The binding of p37 to washed human platelets from 4 normal subjects and two TTP patients after recovery was specific, concentration dependent and saturable. The Scatchard analysis revealed that the binding sites for p37 was about 100,000 per platelet with a dissociation constant of 48 × 10−9 M. The binding of p37 to erythrocytes was very little and non-specific. Stimulation of platelets by thrombin or ADP did not have any effect on the binding of p37 to platelets. The monoclonal antibodies to platelet GP lb (6D1) and GP Ilb-llla (10E5)(A gift of Dr. Barry coller) did not inhibit the binding of p37 to platelets. Fibrinogen (1 mg/ml) and FVIII/vWF (250 ug/ml) reduced the binding slightly. The polyclonal antibodies to p37 as well as concanavalin-A inhibited the binding of p37 to platelets through their direct interaction with p37. Other lectins such as phytohemagglutinin, potato lectin and helix pomatia lectin did not have any effect. At 40 mM, sialic acid, α-D-(+)-glucose, D-(+)-mannose and D-fructose caused 91%,44%,79%, and 63% inhibition of p37 binding respectively. D-(+)-galactose did not interfere with the binding. It is concluded that p37 binds to platelets on the sites other than GP lb and Gp IIb-IIIa and its binding to platelets is inhibited by certain sugars.
PATHOPHYSIOLOGY OF THROMBOCYTOPENIA AND RESULTANT CLINICAL INDICATIONS FOR PLATELET TRANSFUSION
Authors: Slichter Sherrill J
Published: August 2018

Careful evaluation of platelet survival data in normal individuals and patients with thrombocytopeniasecondary to marrow aplasia has demonstrated that platelets are lost from circulation by two mechanisms a fixed fraction of platelets, amounting to approxi mately 7,100 platelets/ul/day, are lost randomly while the remaining platelets are removed by senescent mechanisms. At platelet counts of <100,000/ul, platelet survival becomes progressively shorter as the fixed platelet loss becomes a proportionately greater fraction of the circulating platelets. Thus, there is a direct relationship between platelet count and platelet survival in thrombocytopenic patients. Therefore, when judging the effectiveness of platelet therapy in thrombocytopenic patients, the influence of platelet count on platelet survival must be considered. As yet, there have been no studies to determine if there are ways to interrupt this fixed platelet loss? whether such therapy might improve platelet support in thrombocyotpenic patients by prolonging platelet survival? or, alternatively, whether such therapy might enhance the bleeding risk if random platelet removal is related to physiologic platelet-endothelial cell interactions.

Besides taking into account the effect of thrombocytopenia on the expected response to platelet transfusions, the risk of alloimmunization with platelet transfusion therapy requires a careful assessment of the indications for platelet transfusions for each patient.Based on 51Cr-labeled stool blood loss measurements, we have determined that the bleeding risk is minimal at platelet counts above 10,000 platelets/ul.Only when the platelet count falls to a lower level of 5,000/ul is GI bleeding significantly increased. However, there are certain medications that may enhance the bleeding risk and require platelet transfusions to be given at higher platelet counts.

In those patients who are thrombocytopenic, not because of failure of marrow platelet production, but rather because of accelerated platelet removal, indications for platelet transfusions must be adjusted to meet the particular problem. For example, for patients with autoimmune thrombocytopenic purpura, platelet transfusions are rarely indicated (one exception being intracerebral bleeding) because of the rapid rate of platelet removal and because the patients are usually releasing young hyperfunctional platelets from the bone marrow reducing the hemorrhagic risk at any given platelet count. In some patients with consumptive coagulopathies, even though platelet removal is rapid, platelets may have to be provided until specific therapy resolves the underlying disease process causing the platelet consumption. For these patients, increased levels of fibrinogen/fibrin degregation products, as well as various medications they maybe receiving, may produce platelet dysfunction necessitating platelet transfusions at higher platelet levels. Finally, massive transfusion patients may develop a dilution thrombocytopenia requiring platelet transfusions.
PLATELET TRANSFUSION THERAPY - FUTURE TRENDS AND FEASIBLE GOALS
Authors: Smit Sibinga C Th
Published: August 2018

It is better to know than not to know the devil. Platelet transfusion therapy is one such devil. While the therapy initially is a benefit and might even be life-saving, it often implicates future problems which prohibit optimal efficacy.

The factors determining both the beneficial and the prohibitive effects are complex and not seldom masked. Problem areas include the logistics of availability, the prevention of immunological effects and incompatibilities, the prevention of trans- missable diseases and the prediction of clinical efficacy.Efforts for optimising platelet transfusion therapy should focus on safety, purity, potency and clinical efficacy. However, each of these premises has to be outlined with precision in order to allow targetted development.

Major areas of attention will be the selection and utilisation of available resources, the options for purification, efforts to optimise processing, preservation and storage conditions, innovation of the in vitro viability concept and alternative approaches to selection in order to increase the predictive value of compatibility testing.
SPORADISM INVESTIGATION AND CARRIER DETECTION IN HAEMOPHILIA A BY RFLP ANALYSIS
Authors: Bernardi F , Marchetti G , Vannini F , Felloni L , Panicucci F , Conconi F
Published: August 2018

Restriction fragment lenght polymorphisms (RFLPs)analysis has been employed for carrier detection andfor sporadism study in Haemophilia A. Three RFLPs, one intragenic in FC8 (647/BcII) and two with close linkage to Haemophilia A at DXS52 (Stl4/Taql) and DXS15 (DX13/BgIII), were used.

In 20 families 29 carrier status determinations havebeen performed.

In order to investigate sporadicity and to estimate the sex ratio of mutation rates directely, 17 families with isolated cases of haemophilia A were studied.In eight out of the 17 families the RFLPsanalysis excluded the carrier status of the maternalgrandmothers.Since by hemostatic studies the eight mothers of the propositi were shown to be haemophilia carriers, the origin of the newly mutated genes was inferred from the RFLP patterns: six haemophilic genes derive from the normal maternal grandfathers and two from the maternal grandmothers.Possible recombinations between FVIII locus and the extragenic RFLPs loci have to be considered; however the intragenic Bell RFLP is informative in five out of the eight families and the DXl3 and Stl4 patterns are concordant.

The data indicate a higher mutation rate in males than in females gametes as previously suggested, althought not unanimously, by segregation analysis and coagulation studies. The RFLP analysis in a large number of families with isolated cases of haemophilia isnecessary to define the precise ratio of sex mutation rate for this disease.

Work supported by P.F. Ing. Gen. Basi Mol. Mai. Ered. Contratto CNR N 8400877.
NEW CHROMOGENIC SUBSTRATES FOR THE DETERMINATION OF COAGULATION AND FIBRINOLYSIS ENZYMES
Authors: Stüber W , Schiwek D , Becker U , Heimburger N
Published: August 2018

A new type of chromogenic substraces based on derivatives of phenoxazine is presented. Particularly interesting is the blue dye 7-amino-3-diethylamino-8-methylphenoxazine (ADMP) with a molar extinktion coefficient of about 80,000 at 624 nm. Peptides were linked to the aminogroup of the dye and red coloured substrates were obtained with a λmax value of about 540 nm. On account of the distinct difference of the λmax values and the negligable influence of the absorption peaks of the acylated and the free dye this chromophore is suitable for the synchesis of substrates. Besides the spectral properties of chese new chromogenic peptides we determined their characce-riscics using serine proteases involved in the process of coagu-lacion and fibrinolysis. In comparison co para-nitroaniline substrates the introduction of the relatively bulky ADMP into the peptide sequence led to products with superior properties in respect of sensitivity and specificity.

It was found that the ADMP substrates are particularly favourable for the determination of thrombin, urokinase and activated protein C in the presence of other proteases.
CONTINUOUS PERIOPERATIVE ADMINISTRATION OF Cl-ESTERASE-INHIBITOR CONCENTRATE AND APROTININ IN CARDIOVASCULAR SURGERY
Authors: Poppv S , Philapitsch A , Murday H , Fenyes J , Kirchhoff P G
Published: August 2018

The results obtained in our randomised study in 3 groups of patients performed in 1982/83 suggested the administration of protease inhibitors to be effective in counteracting the activation of the kallikrein, coagulation and fibrinolytic systems during extracorporeal circulation (ECC) thus lowering the rate of postoperative bleeding complications. Since Cl-esterase-inhibitor (Cl-INH) proved to be the main inhibitor of the kallikrein (KK) system and aprotinin that of plasmin further studies to investigate the efficacy of a combined treatment and to develop an improved dosage regimen were initiated in 1985.

Pilot:1: 3 patients/aprotinin; pilot 2: 3 patients/Cl-INH Concentrate; pilot 3: 5 patients/Cl-INH Concentrate + aprotinin. Test parameters included: protein/Biuret; Cl-INH/rocket immune electrophoresis and chromogenic substrate method; kaolin induced kallikrein generation; plasminogen activation using streptokinase; plasmin inhibiton capacity; coagulation factors I, II, V, VII, VIII, and X; reptilase clotting time, thrombin time; platelets and the thrombelastogram.

15 blood samples were taken at regular intervals over approx. 16 hours before, during and after surgery, as well as on the first and second postoperative day.

Dosage in pilot 1 and 2 was 500 × 103 KIU aprotinin and 1500 PU Cl-INH, respectively, followed by the continous i.v. infusion of 125 × 103 KIU aprotinin/h and 125 PU CI-INH/h, respectively over approx. 16 hours. This regimen still appeared inadequate to prevent the activation of the KK and fibrinolytic system. In pilot 3 therefore, the continous infusion of both concentrates was increased by 100 % (250 × 103 KIU aprotinin/h + 250 PU Cl-INH/h), while the bolus-injection remained the same as in pilot 1 and 2. Using this optimised combined regimen parameters of the kallikrein, coagulation and fibrinolytic system remained almost unchanged compared to values obtained prior to ECC.
ORNITHO-KININOGEN: ITS ISOLATION AND CHARACTERIZATION
Authors: Kimura M , Sueyoshi T , Morita T , Tanaka K , Iwanaga S
Published: August 2018

In mammals, three kinds of kininogens, high-Mr kininogen, low-Mr kininogen and T-kininogen, are known and their structures and functions have been well studied. However, little is known about non-mammalian kininogens and their existence has been known only by the release of plasma kinins. In 1966, Werle et al. reported that an ornitho-kinin liberated by tissue kallikrein from chicken, plasma differs;from mammalian kinins in its amino acid composition. Since that, there is no report about the structure of ornitho-kinin.

In this study, ornitho-kininogen was isolated from chicken plasma by two steps with S-alkylated papain affinity and DEAE-5PW columns. The yield was 1.7 mg from 44 ml of plasma. The purified material gave a single band on SDS-PAGE with or without 2-mercaptoethanoL and on disc-PAGE, and the molecular weight was estimated to be 74,000 on SDS-PAGE by Ferguson plot. Ornitho-kininogen seemed to belong to mammalian high-Mr kininogen, based on the amino acid composition, and the molecular weight, and no kininogen corresponding to low-Mr kininogen and T kininogen was found in chicken plasma. In fact, ornitho-kininogen was degraded rapidly by bovine plasma kallikrein, liberating an ornitho-kinin, and the resulting kinin was separated by reversed phase HPLC on a column of Cosmosil 5C18-P. The amino acid sequence of ornitho-kinin was established to be Arg-Pro-Pro-Gly-Phe-Thr-Pro-Leu-Arg, in which the composition was different from that reported earlier by Werle et al. This sequence was similar to that of bradykinin but the two substitutions of Thr-6 and Leu-8 for Ser and Phe were found. Ornitho-kinin induced a hypotension on chicken and contracted the isolated smooth muscle. However, it did not any effect on the isolated rat uterus, suggesting that the specificity may be due to replacement of Phe-8 by Leu. The NH2-terminal 30 amino acid residues of ornitho-kininogen exhibited 43 % identity with that of bovine kininogen.
EFFECTS OF PLASMA KALLIKREIN AND BRADYKININ ON FIBRINOLYSIS AND THROMBOXANE PROSTACYKLIN FORMATION STUDIED IN MINIPIGS
Authors: Egberg Nils , Gréen Krister , Jacobsson Jan , Vester-gvist Ole , Wiman Bjöm , Gallimore Michael
Published: August 2018

The effect of plasma kallikrein and bradykinin infusions into pigs on hemodynamic and hemostatic variables have been investigated. Both substances caused a pronounced decrease of the systemic blood pressure. The leucocyte count in periferal blood fell markedly, reaching a minimun within one hour after infusion of either of the substances.

Signs that could be interpreted as a progressive disseminated intravascular coagulation with decrease of fibrinogen and platelet count was observed after kallikrein as well as bradykinin infusions. A pronounced increase of the plasma tissue plasminogen activator concentration followed both plasma kallikrein and bradykinin infusions. However, the peak concentration was found 5 minutes after bradykinin infusion but 60-120 minutes after kallikrein infusion, suggesting different mechanisms leading to the t-PA release. Concomittant with the maximun t-PA concentration there was a marked reduction of the plasminogen activator inhibitor (PAI) concentration. Three hours after drug infusions the PAI concentration was at or above preinfusion level. Kallikrein infusions caused a 10-20 fold increase of the urinary excretion of 2,3-dinor thromboxane B2 (metabolite of TxA2) and a 3-42 fold increase of 2,3-dinor-6-keto-PGFlalpha (metabolite of PGI2) excretion respectively. Corresponding data for bradykinin infusions were, 1.6-5 fold and 2-10 fold increases respectively. Possible links between leucocyte aggregation, prostanoid formation and fibrinolytic variables will be discussed.
PERTURBATION OF THE KALLIKREIN-KININ SYSTEM IN ADULT RESPIRATORY DISTRESS SYNDROME (ARDS)
Authors: Walshe K , Mackie I , Gallimore M , Machin S J
Published: August 2018

Platelet and fibrin deposition in the small blood vessels of the lung as well as activation of the contact system with consequent kinin generation have been described in ARDS. It is thought that these haemostatic changes may play a role in the pathogenesis of the disease which include dyspnoea, hypoxaemia, pulmonary oedema, and pulmonary hypertension. We have studied a complete battery of tests for the proteases and inhibitors of the kallikrein-kinin system in an ongoing study of ARDS and haemostasis. 12 patients have so far been studied, with the following factors complicating ARDS: 5 had sepsis, 3 post surgical, 1 head injury, chronic renal and airways disease, renal transplant rejection, or after smoke inhalation during a fire. Assays for: FXI, FXII, prekallikrein (PKK), kallikrein inhibitor (KKi), betaFXIIa inhibitor, (BXIIAi), alpha-2-macroglobulin (α-2-M), alpha-l-antitrypsin (α-1-AT), and antithrombin III (ATIII) were performed by microtitre amidolytic assays to allow the economic processing of large numbers of samples. FXII, PKK, ATIII and α-2-M were invariably low, returning towards normal as the patient became clinically well. The PKK level in particular reflected the clinical course of the patient and appeared to have prognostic significance. Surprisingly, the BXIIai and KKi tended to be increased when the FXII and PKK were decreased. FXI and -1-AT levels showed no change. These results suggest that clinical concentrates of haemostatic inhibitors may be of benefit in ARDS, to stop the continuous activation of the kallikrein-kinin system and generation of biologically active peptides.
SERUM VITAMIN K1 LEVELS AS AN EARLY INDICATOR OF HYPOPROTHROMBINAEMIA ASSOCIATED WITH ANTIBIOTIC THERAPY
Authors: Machin S J , Cohen H , Mackie I J , Shearer M , Scott S D
Published: August 2018

The prothrombin time is an insensitive indicator of early vitamin K deficiency and serum vitamin K1 levels may correlate with liver stores. A random non-fasting range of serum vitamin K1 was established in 45 healthy adults of 150-1,530 pg/ml (mean 412 pg/ml). Nine well nourished patients, with normal serum vitamin K1 levels, (mean 546, range 310-1,350 pg/ml), maintained normal prothrombin times and factor VII clotting activity throughout 7 days therapy with cefotetan disodium, an NMTT-containing cephalosporin antibiotic. However, 11 of 20 patients with acute intra-abdominal sepsis and an initially normal prothrombin time who underwent emergency surgery, developed a raised prothrombin time (INR 1.4-3.1) associated with reduction in factor VII activity (0.74 to 0.38 iu/ml) after 3-7 days of antibiotic therapy and the presence of PIVKA II by crossed-immunoelectrophoresis. Nine of these 11 patients had clinical evidence of malnutrition by anthropometric assessment and subnormal serum vitamin K1 (mean 119, range 43-354 pg/ml) levels on admission. Seven received cefotetan but 4 were treated with other non-NMTT containing antibiotics. The 9 patients who maintained normal prothrombin times and factor VII levels had normal nutritional status and normal serum vitamin K1 levels (mean 279, range 103-915 pg/ml) at presentation. A low serum vitamin K1 level was associated with a high incidence of hypopro-thrombinaemia developing following antibiotic therapy and would appear a more sensitive indicator of reduced vitamin K stores than the prothrombin time.
PROTECTIVE EFFECT OF VITAMIN K AGAINST ACETAMINOPHEN (PARACETAMOL) TOXICITY IN THE HAMSTER
Authors: Malia R G , Kennedy H J , Triger D R , Preston F E
Published: August 2018

We have previously reported that paracetamol may interfere with the metabolism of vitamin K via the vitamin K 2, 3-epoxide cycle. (Thrombosis and Haemostasis, 54,205,1985). In this study we have examined the effects of large doses of vitamin K on experimentally induced paracetamol hepatic necrosis in a hamster model. Paracetamol (1.2g/Kg) when given by gavage to 24 hamsters (Group I) resulted in 10 deaths (42%) at 24 hours. Simultaneous administration of lmg vitamin K intraperitoneally (Group II) reduced mortality to 3/24 (12%); mortality was 2/24 (8%) if vitamin K was given 4 hours after the paracetamol (Group III). In a further series of experiments (N=18) the prothrombin time in Group I was prolonged by 28 seconds compared with 14 seconds in Group II and 10 seconds in Group III. In samples taken for biochemical and histological analysis there was evidence of severe hepatic necrosis in all groups. When the dose of paracetamol was reduced to 1.0g/Kg (N=18) there was substantially less histological damage in Group II compared with other groups and the prothrombin times were only prolonged by 2 seconds (Group I); 1.5 seconds (Group II) and 3-5 seconds (Group III) respectively. In the final experiment (N=18) when Group II animals were divided into 3 sub-groups and the dose of vitamin K given was altered to 1.0, 0.5 and O.lmg following the paracetamol (1.2g/Kg) the prothrombin times were prolonged by 8 seconds, (1-0mg vitamin K), 21 seconds (0.5mg vitamin K) and 35 seconds (0.1mg vitamin K) respectively, indicating a dose dependant effect. The hypothesis that paracetamol induced hepatic necrosis occurs solely as a consequence of failure of glutathione to conjugate the reactive metabolites of paracetamol is not consistent with the protective effects of vitamin K observed here and the known mode of action of the vitamin. Other mechanisms such as free radical scavenging deserve to be studied.
EFFECTS OF GABEXATE MESILATE (FOY), A NEW SYNTHETIC SERINE PROTEASE INHIBITOR, ON BLOOD COAGULATION IN PATIENTS WITH DIC
Authors: Palareti G , Maccaferri M , Poggi M , Petrini F , Coccheri S , Haverkate F , Montanari F , Corticelli A S
Published: August 2018

A pilot open controlled study of FOY was performed in 20 intensive care patients (pts, age 18-63) with DIC diagnosed with standard laboratory criteria (at least 3 of the following: Normotest 70%, fibrinogen 150 mg%, AT III 80%, FDP 20 ug/ml, platelets 150000). Besides the usual treatments, FOY was given to 10 pts (FOY G.) by continuous i.v. infusion (1mg/kg/h) for up to 7 days, while in 10 control pts (Hep.G.) the treatment included low dose s.c. heparin. Blood clotting tests were performed at admission to the study and daily for 7 days; we consider here results obtained at baseline and at the 4th (7 survivors in FOY G. and 10 in Hep.G.) and the 7th day (6 surv. in FOY G. and 9 in Hep. G.). Statistical evaluation was made by means of the twotailed Wilcoxon test for non parametric paired data. In the FOY G. depressed baseline AT III and plasminogen (Plgn) activities (61.8+/-5.3% and 57+/-5.5% respectively) significantly increased at 4th day (92+/-11.2% and 83+/-3.1% p<0.05), Plgn furtherly significantly increased at 7th d. (p<0.05). In the Hep.G. Normotest, Plgn and Platelets significantly (p<0.05) increased at 4th day, but no changes in AT III were found. Fibrinogen increased in both groups during the observation period (pA0.05). Serum and especially "plasma" FDP (specific monoclonal Ab to fgn and fibrin degradation products), as well as D-Dirner and HMW fgn complexes measured by exclusion chromatography, decreased especially in the FOY G. These results show that FOY modifies the clotting laboratory pattern in DIC pts, likely by an antithrombin effect. The clinical significance of these effects remains, however, to be explored.
INHERENT DANGERS OF SIMULTANEOUS APPLICATION OF MICROFIBRILLAR COLLAGEN HEMOSTAT AND BLOOD-SAVING DEVICES
Authors: Robicsek F , Duncan G D , Born G V R , Wilkinson H A , Masters T N , McClure M
Published: August 2018

Microfibrillar collagen hemostat (MCH) is a topically applicable hemostatic agent which has been introduced in clinical practice recently and is marketed under different trade names and recommended for use in the course of surgical procedures. Because of the possibility that this substanee may pass through different blood-collecting circuits and cause organ damage if reintroduced into the patient’s circulation, a series of in vitro and in vivo experiments were performed. In the course of in vivo experiments the possible passage of MCH through "cell saver" devices was investigated. In Group A the passage of 1-125 labeled MCH was injected through different parts of a "cell saver" circuit. In Group B experiments the same was studied through a passage of 40μum pore-size blood transfusion filters. In the in vivo experiments MCH was injected intravenously into different parts of the' circulatory system of rabbits after different degrees of dilution and filtering after which the animals were killed at different time intervals and the radioactivity was determined in respective brain sections by microradiography. The results of these experiments suggest that the passage of MCH indeed occurs through different blood collecting and filtering devices and results in direct embolization of vital organs. This cannot be prevented even with the application of commercially used filters. Therefore, the authors recommend that the substance should not be used if shed blood is intended to be collected and returned to the patients circulation.
HEPARIN COFACTOR II ASSAY : IMPORTANCE AND CONTROL OF THE NON-CONTAMINATION BY HEPARIN OF DERMATAN SULFATE
Authors: Tapon-Bretaudière J , Fischer A M , Millien C , Bros A
Published: August 2018

A chromogenic substrate method for the determination of heparin cofactor II (HC II) in plasma, based on its anti Fc Ila activity, has been developped in our laboratory, using dermatan sulfate (D.S.) as activator. We observed that D.S. from porcine skin, which was claimed not to be contaminated by heparin, was sometimes contaminated, depending upon the batch used. Moreover, a classical treatment of D.S. by nitrous acid, that specifically degrades heparin-like glycosaminoglycans, is not always sufficient and in some cases, needs to be repeated. It is thus important to check the absence of heparin contamination for the validity of the HC II assay. Indeed, we have shown that for an amount of contaminating heparin as small as 5×10−5 iu per μg of D.S., the HC II level of a normal plasma was increased by 50%. Such a low contamination is undetected by routine tests (thrombin time, APTT, heparin activity assay). Thus, we propose two useful methods for this purpose : (i) an anti-Fc Xa heparin cofactor activity assay on normal plasma, using D.S. as activator ; HC II being devoid of any anti-Fc Xa activity, if a Fc Xa inactivation is observed, it is due to AT III potentiation by the contaminating heparin, (ii) an HC II assay using a normal plasma and an AT III depleted plasma ; in the case of D.S. contamination, a higher level of HC II is obtained with normal plasma, reflecting the AT III potentiation by heparin. For example, with 2.5×10−5 iu of heparin per μg of D.S. : (i) an anti-Fc Xa activity of 50% was found, decreasing to 0% after decontamination, (ii) the HC II. level which was 125% in normal plasma, compared to 100% in AT III depleted plasma was identical (100%) in both plasmas after decontamination. These results demonstrate the validity of the two methods and the usefulness of a control of each batch of D.S. used in HC II assay, even after nitrous acid treatment of the glycosaminoglycan.
KINETICS OF HEPARIN CO-FACTOR II (HCII):THROMBIN AND ANTITHROMBIN III (ATIII):THROMBIN INTERACTION. MOLECULAR WEIGHT DEPENDENCY
Authors: Ellis V E , Scully M F , Kakkar V V
Published: August 2018

The influence of increasing concentrations of heparin of different molecular mass (Mr) has been compared in potentiation of the rate of HCII:thrombin interaction and of ATIII:thrombin interaction under pseudo first order conditions. Unfractionated and fractionated heparin showed a concentration dependent ascending and descending limb of stimulation of the rate which was closely similar for both inhibitors. Unfractionated heparin and fractions of 16.5 KDa or less showed a peak acceleration of the rate of interaction of thrombin with both inhibitors at 0.3×10−6 heparin although the observed maximum rate at this peak decreased with fall in Mr. For both inhibitors two high Mr fractions (22KDa and 32KDa) showed peak stimulation at a lower heparin concentration (0.3×10−7M) and 1.5 to 2 fold greater increase in rate than that observed with unfractionated heparin. Under these conditions it could be calculated that the potency of a 32KDa fraction was 1200iu/mg with respect to UF heparin (150iu/mg). Three further pools were prepared and ATIII .high affinity fraction prepared by chromatography. Acceleration of rate of interaction was measured according to concentration and inverse plots gave values for apparent Kd amd maximal rate.

These results suggest that differences in the profiles of stimulation by high Mr fractions to those of lower Mr are related to higher binding affinities for the inhibitor permitting maximal binding of heparin before the descending part of the slope due to saturation of thrombin (according to the template hypothesis). Although close similarity was found between heparin stimulation of HCII and ATIII, potentiation of HCII inhibitory activity differed in that it was reversed by lower ionic strength and was not reversed by a heparin pentasaccharide with high affinity for ATIII.
EFFECT OF THE BUFFER COMPOSITION AND THE PRESENCE OF ORG 10172 AND SOME GLYCOSAMINOGLYCANS ON THE INHIBITION OF ALPHA-THROMBIN BY HEPARIN COFACTOR II
Authors: Verhamme I M A , van Dedem G W K
Published: August 2018

Heparin cofactor II (HCII) inhibits alpha-thrombin (Ila) with formation of a 1:1 complex.The inhibition is accelerated by heparin and dermatan sulfate (DS).The present study describes the pH and ionic strength dependence of the second order inactivation constant k2nd for the simple IIa-HCII interaction and for the interaction catalysed by heparin, dermatan sulfate and the antithrombotic Org 10172 containing low molecular DS.In all cases we found a bell-shaped pH profile for k2nd with characteristic maxima and pK values. Org 10172 accelerates the HCII mediated inhibition of Ila though not to the same extent as heparin and DS. Substantial narrowing of the pH profile occurs under influence of glycosaminoglycans , the alkaline limb of the profiles being displaced towards a more acidic pH. The ionic strength is critical for the determination of k2nd. A nonlinear decrease is observed with increasing ionic strength, due to the screening effect of the ions in solution. Ca++ ions do not accelerate the inactivation process in absence of GAG, in contrast to the findings for the Ila-ATIII interaction. The pseudo-first order model for Ila inactivation is only valid for : a) the uncatalysed reaction irrespective of ionic strength, b) the inactivation in buffers of intermediate ionic strength and in the presence of low GAG concentration and c) the inactivation in media of high ionic strength irrespective of GAG concentration. In all other reaction conditions the inactivation rate substantially deviates from a pseudo-first order plot.Suicide substrates are known to exhibit a comparable behavior. However this model appeared inadequate for describing our experiments. An appropriate model for these aberrant inactivation profiles is not evident.Deviation from pseudo-first order kinetics occurs more readily with heparin than with dermatan sulfate and is probably related with their difference in charge density.
AN INDIRECT KINETIC METHOD FOR ESTIMATING THE AFFINITY BETWEEN HEPARIN AND HEPARIN COFACTOR II
Authors: Visser A , Verhamme I M A , Meuleman D G , van Dedem G W K
Published: August 2018

The heparin-dependent inactivation of alpha-thrombin by heparin cofactor II was studied in buffer media with a pH ranging from 6 to 9 and an ionic strength from 0.05 M to 0.80 M. We used a heparin fraction with a mean Mr of 16,000 .The log dose response curves (logarithm of the 2nd order inactivation constant vs. the logarithm of heparin concentration) display a sigmoidal behavior. The lower and upper limiting plateau and the steepness of the ascending limb are characteristic for every pH and ionic strength. Similar log dose response curves can be observed for the heparin-mediated inactivation of factor Xa and plasmin by ATIII,indicating that enhancement of the inhibition only depends on heparin-inhibitor binding despite the presence of heparin-enzyme complexes.This type of inactivation mechanism is clearly different from the one observed for the thrombin- and factor IXa - ATIII interactions which are characterized by a maximum in the log dose response curves.Therefore we can make the assumption that heparin-inhibitor binding is of major importance in the heparin-mediated thrombin-HCII interaction.Our experimental data were fit to a model which describes the dependence of the observed inactivation constant upon the concentration of heparin-HCII complex.This complex concentration is a function of the initial heparin and HCII concentrations and the dissociation constant K0 of the heparin-HCII complex.The model allows the estimation of K0 in various buffer media.A decrease of pK0 with increasing buffer concentration can be observed.The upper limiting plateau value for kobs which is often referred to as the intrinsic activity of heparin also decreases with increasing ionic strength.At pH 7 and 8 and an ionic strength of 0.4 M we-found K0 values of 1.00E-07 M.At pH 6 and an ionic strength of 0.8 M Kq eguals 4.00E-04 M indicating a markedly decreased affinity of heparin for HCII.Through a detailed analysis of the pH profile for K0 we might gain insight in the nature of the binding sites for heparin on the inhibitor.
EFFECT OF VARIOUS HEPARIN(OID)S ON HEPARIN COFACTOR II MEDIATED ANTI-THROMBIN ACTIVITY AND INHIBITION OF THROMBIN GENERATION IN VITRO
Authors: van Dinther T G , Hol F , Meuleman D G
Published: August 2018

The effects of various heparin(oid)s, standard heparin VII (SH), dermatan sulphate (DS), a low molecular weight fraction of heparin (UMW-H), FragminR (FRA), Org 10172 = low molecular weight heparinoid, the fraction of Org 10172 with high affinity for AT-III (HA-10172) and the low affinity fraction (LA-10172) respectively were examined on in vitro thrombin generation and inactivation.

Thrombin inactivation in the presence of either heparin cofactor II (HC-II) or anti-thrombin III (AT-III) was assessed with two newly developed assays using the purified cofactors, thrombin and chromogenic substrate S2238 on microtiterplates. Thrombin generation in the presence of HC-II and AT-III was studied using purified factor Xa, prothrombin and blood platelet lysate and the residual thrombin activity was assessed amidolytically.

The inhibition of the compounds on thrombin activity are summarized in the table

The following conclusions can be drawn:

- SH, LMW-H, HA-10172 and FRA potentiate the AT-III mediated inactivation of Ha more strongly than the HC-II mediated inactivation.

- DS and LA-10172 show the reverse pattern of inactivation, while Org 10172 potentiates both inactivaton pathways to a similar extent.

Thrombin generation in the presence of HC-II is inhibited by mw-heparin(oid)s at approx. 2-5 times lower concentrations than the HC-II mediated thrombin inactivation, while the inhibiting effect of SH in both assays is comparable.

AT-III mediated thrombin generation inhibition and AT-III mediated thrombin inactivation is comparable as well for SH, LMW-H and FRA. In contrast, Org 10172 and its subfractions are approx. 10 times more potent on AT-III mediated thrombin generation inhibition than on AT-III mediated thrombin inactivation.

Org 10172 shows low anti-thrombin activity and this activity is mainly mediated via FC-II.
IN VITRO REACTION MODELS OF THROMBIN AND ITS PHYSIOLOGICAL INHIBITOR ANTITHROMBIN III IN THE PRESENCE OF HEPARIN
Authors: Niederst P N , Asbach M , Ott M , Zimmermann R E
Published: August 2018

Antithrombin III (AT III) neutralizes thrombin and other serine proteases of plasma coagulation system by forming a stable 1:1 covalent complex. The inhibition rates are greatly increased by the potent catalyst heparin. The catalytic mechanism of heparin was studied in the presence of dextran sulfate (DS), a thrombin-binding sulfated Polysaccharid. DS did not influence the reaction of AT III with heparin and the amidolytic activity of thrombin, but preincubation with thrombin could inhibit the catalytic activity of heparin in the reaction of thrombin with AT III. We conclude that the reaction of heparin with enzyme and inhibitor, thus forming a ternary complex, is necessary for its catalytic activity.

It is known that heparin also converts AT III from an inhibitor to a substrate for thrombin in a dose dependent manner. By cleavage of the reaction site bound Arg(385)-Ser(386) an AT III-fragment (MG 50000 d) occurs, which has a decreased affinity to heparin and does not inhibit F I la. At physiological ionic strength we have only measured a small percentage of AT 111-proteolysis (4%, 1 U/ml Hep). The extent of AT III-fragment formation could be enhanced by lowering the ionic strength (max 44%, 1 U/ml Hep., 1=0,02).
THE ROLE OF HEPARIN CHARGE DENSITY IN THE ANTITHROMBIN III-DEPENDENT AND ANTITHROMBIN III-INDEPENDENT INACTIVATION OF THROMBIN
Authors: Baruch D , Franssen J , Hemker H C , Lindhout T
Published: August 2018

The dependence of the anticoagulant properties of heparin upon charge density may reflect structural factors that are important in anti-thrombin effect. We have previously demonstrated that in the absence of antithrombin III (AT III) unfractionated heparin inhibits the catalytic effect of thrombin upon platelet activation. In the present study we evaluated the thrombin-binding properties of heparin fractions obtained by ion-exchange chromatography on DEAE-Sephacei. We found that these fractions were able to bind to thrombin with an affinity that increased with their charge density. This was shown by their inhibitory effect in the absence of AT III on thrombin-catalyzed platelet factor Va formation and by the ability of active site blocked thrombin to prevent the heparin-dependent inactivation of thrombin by AT III. However, their increase in charge density and thus affinity for thrombin was found to go along with an increase in AT III-binding sites, as measured by the heparin-dependent increase of the intrinsic fluorescence of AT III. Moreover all heparin fractions showed the same specific antithrombin activity when the molar concentration of AT III-binding heparin was taken into account. We also investigated the thrombin-binding properties of two heparin fractions obtained by affinity chromatography on AT III-Sepharose. The AT III low affinity fraction was practically devoid of any inhibitory effect on the rate of the thrombin-catalyzed factor Va formation, indicating a low, if any, affinity for thrombin. In contrast the AT III-independent inhibition of thrombin was completely recovered from the AT III high affinity fraction. In addition, we also established that when the heparin fraction from the DEAE-Sephacel column, with the lowest charge density and very low in AT III binding material, was modified by the incorporation of sulfate groups so as to achieve a higher charge density, it obtained a higher affinity for thrombin but this modification caused the loss of half the AT III binding sites. In conclusion, it is apparent that fractionation of crude heparin on a DEAE-Sephacel column or on an AT III-Sepharose column does not result exclusively in a separation of either the thrombin-binding or the AT III-binding heparin fractions.
ANTITHROMBIN III AND HEPARIN COFACTOR II IN PATIENTS WITH CHRONIC RENAL FAILURE
Authors: Toulon P , Jacquot C , Frydman M O , Vignon D , Aiach M
Published: August 2018

Anti thrombin III (AT III) and heparin cofactor II (HC II) were measured in 77 blood donors, 82 patients with chronic renal failure (CRF) undergoing regular hemodialysis and 36 undialyzed patients with CRF. AT III was measured as heparin cofactor and HC II as dermatan sulfate cofactor using amidolytic assays.

The results (mean ± SD expressed in percentage of the in pooled normal plasma) are summurized in the table.

Subnormal AT III levels were found in both dialyzed and undialyzed patients with CRF, while HC II was significantly (p < 0.001) reduced in dialyzed patients (12 of them were found to have HC II levels below to lowest value founded in our control group: 68 %).

In order to explain this decrease of HC II level in dialyzed patients with CRF, we compared both AT III and HC II activities before and after a dialysis session in 24 patients (12 with a low and 12 with normal before dialysis HC II activity). AT III and HC II increased significantly (p < 0.01) in all patients after dialysis. When related to total plasma proteins in order to suppress the influence of hemoconcentration induced by dialysis, AT III decreased significantly (p < 0.01) in the 24 patients while HC II increased significantly (p < 0.01) only in patients with low before dialysis HC II levels (the increase in HC II activity was found significantly in the pooled 24 patients).
STUDIES ON LAL INHIBITORY REACTION BY HUMAN SERUM ALBUMIN AND ATIII PRODUCT
Authors: Homma R , Miura Y
Published: August 2018

Test for pyrogenicity in human blood products using rabbit has been authorized as one of the security test, but LAL ( Limu-lus Amebocyte Lysate ) test has not been approved in Japan. We have studied the problem by using the in vitro LAL test ( endotoxin test ) for the rabbit pyrogen test of blood products expecially human serum albumin ( commercial HSA and PPF ) or ATIII products. It is well known that the LAL inhibitory reaction has been detected, although slightly, in most human serum albumin products, and therefore, the factors participating in this inhibition were investigated using commercial HSA and PPF products, and also preparations without heating in their production process. ATIII activity was assayed by two methods clotting and chromogenic substrate method: the LAL activity was assayed by gelation method ( Toxinometer, Wako-Junyaku Kogyo Co.). We found that the LAL activity in lion-heated PPF preparation ( NHPPF ) was determined to be significantly lower than heated PPF preparation ( PPF product ) from one manufactuerer, but not from others. The ATIII activity of this particular NHPPF was extremely high as compared with the other PPF. Addition of ATIII to the PPF caused a significant decrease of the LAL activity in reciprocal proportion to its increase. The LAL inhibitory activity and ATIII activity of the NHPPF were inactivated by heating at 60° C for 2.5 hrs. We also found that ATIII products from the two manufactuerers possessed remarkable LAL inhibitory activity which also induced a decrease by heat treatment at 56° C for 30 min. The ATIII activity was considered to be one of LAL inhibitory factors from the corelation between LAL reactivity and ATIII activity.
PROPHYLAXIS OF THROMBOEMBOLISM DURING PREGNANCY IN THREE SISTERS WITH CONGENITAL ANTITHROMBIN III (AT m) DEFICIENCY COMBINED WITH REDUCED INDUCIBLE FIBRINOLYTIC ACTIVITY
Authors: Schoch U , Zanetti E , von Felten A
Published: August 2018

AT III deficiency is associated with a high risk of venous thromboembolism, particularly in pregnancy. As prophylactic treatment it has been recommended to normalize plasma levels of AT in by use of AT III concentrates, besides giving heparin.

We report on the prophylactic treatment of three sisters (age 21, 25, 32 years) with congenital AT in deficiency (38-53%, normal: 80-120%) as well as a reduced inducible fibrinolytic activity (1.2, 5.8, 7.9%, normal: >8.5%), who already had suffered from severe thromboembolism. During pregnancy prophylactic measures were taken individually, depending on the plasma level of 3-throm-boglobulin (BTG) determined every 2-3 weeks. At the time of the first increase of BTG (around 10th week of gestation) prophylaxis with s. c.heparin 2x7'500IU/d was started, leading to normalization of BTG. When BTG was again elevated, the dose of heparin was successively increased up to 2x15'000IU/d; thereby, functional AT IH levels remained in the range of 28-50%. Two patients received only heparin throughout the pregnancy. However, in one patient BTG levels could not be normalized by heparin alone (60-130ng/ml, normal: <43ng/ml). Injections of AT in concentrate, 11000IU, led to reduction of BTG within 2 hrs (60 → 42, 220 → 61 ng/ml). Therefore, AT m was given from the 25th week of gestation in increasing amounts up to 5'000IU/week (funct. AT m in plasma: 51 -72%) in addition to heparin (2x12'500IU/d), resulting in BTG levels of 33-51 ng/ml. From the onset of labour, all patients received AT IU concentrates (two patients l'OOOIU every second day, one patient 1'0001U daily) together with i. v. heparin 20'000-27'500IU/d, until the oral anticoagulant treatment started after delivery had reached therapeutic levels. The amount of AT m concentrate totally administered was 5'000IU in two patients and 66'000IU in one patient. None of the patients showed ever signs of venous thrombosis.

Our observations demonstrate that instead of an AT in substitution with the aim to normalize its plasma level, an effective thrombosis prophylaxis - monitored by plasma levels of BTG - may be achieved with heparin alone or combined with low amounts of AT in concentrate.
RANDOMIZED TRIAL OF ANTITHROMBIN III VERSUS PLACEBO IN PATIENTS UNDERGOING PERITONEO-VENOUS SHUNT OPERATION
Authors: Korninger C , Klepetko W , Miholic J , Schwarz Ch , Lechner K
Published: August 2018

A randomized trial of Antithrombin III (AT III) versus placebo was performed in patients undergoing peritoneo-venous shunt operation because of intractable ascites. 10 patients with alcoholic liver cirrhosis (Child's stages B and C) were enrolled. Randomization was performed according to the stage of liver disease and to preoperative AT III levels. AT III concentrate (kindly provided by Kabi) was infused in 5 patients, twice daily for 4 days, at a dose of 20 U/kg BW, starting 12 hours prior to operation. Coagulation studies were performed preoperatively, and on postoperative days 1, 3 and 7.

In all patients, ethanol gelation test was positive on postoperative days 1 and 3, indicating the presence of fibrin monomers. No difference between the two groups was seen with respect to prothrombin times, coagulation factor levels, FPA concentrations or fibrinolysis parameters. It is concluded that the severity of disseminated intravascular coagulation can be tempered by AT III substitution, but, with the administered dosage, DIC cannot be prevented.
ANTITHROMBIN III NORTHWICK PARK: CHARACTERIZATION OF AN INACTIVE HIGH MW COMPLEX WITH INCREASED AFFINITY FOR HEPARIN
Authors: Erdjument H , Lane D A , Flynn A M , Ireland H , Panico M , Marzo E V Di , Blench I , Morris H
Published: August 2018

It has been shown previously that antithrombin III Northwick Park (AT III NWP) has reduced ability to inactivate thrombin and is characterised by an additional anodal component on crossed immunoelectrophoresis. We have applied plasma from an affected family member to heparin-Sepharose and eluted the AT III with a salt gradient. Evidence will be presented that the anodal component has* higher affinity for heparin than normal AT III. Furthermore, this variant component is present in plasma as a MW >120,000 inactive complex whose tryptic peptide FAB map contains numerous signals not characteristic of normal AT . 111 . This complex can be reduced with dithiothreitol to two non identical bands on SDS PAGE with MW ~60,000, only one of which reacts with anti-AT III. Using ion-exchange chromatography and HPLC these two components have been isolated and separated. The N-terminal sequence of the protein that does not react with anti-AT III is believed to be Asp-Ala-His-Ile-Ser-Glu. Structural investigations on the variant AT III are underway.