bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement-Dependent Activation Of Plasminogen By Activated Factor B (Bb) Of The Alternative Pathway Of Complement
Authors: Sundsmo J S , Wood L M
Published: July 2018

Activated Factor B (Bb) of the alternative pathway of complement (APC) induces human monocytes and murine macrophages to spread on a glass substrate (Sundsmo and Gątze (1980), Cell. Immunol., 52:1). In studies designed to investigate the effects of Bb on plasminogen activator secretion by human monocytes, it became apparent that Bb possesses innate plasminogen activator (PA) activity. PA activity of Factor Bb was determined by release of radiolabeled fibrin peptides (modified fibrin plate assay) over 4 hrs. at 37°C using purified human plasminogen, 125I-labeled human fibrinogen, and 20% fresh normal human serum as a source of thrombin. Native Factor B did not express significant PA activity, however, purified Bb, or Bb in complex with cobra venom factor released, 52±8% and 79±4%, respectively, of the 125I- fibrin that is released by urokinase. Intermediate complement complexes on rabbit erythrocytes (Er) were tested for their PA activity: ErC3b, Bb released 76±7% of 125I-fibrin, and control Er, C3, and B released <15%. Factor D was without activity. Cleavage of plasminogen by Factor B was investigated by incubating cellular complement intermediates (ErC3b, Bb; EsC3b, Bb, NF) 30 min./37°C with 125iI-labeled plasminogen; and, conducting SDS-PAGE (reducing conditions) to separate native plasminogen from the heavy and light chains of plasmin. It was found that Bb cleaves predominantly the Lys-form of plasminogen to generate fragments with apparent molecular weights of 72,000 and 30,000. As a control, 125I-labeled plasminogen was incubated with Er, C3, or D, and <10% cleavage was observed; EsC3b or native Factor B cleaved <5%. Affinity-purified goat anti-B Ig inhibited Factor Bb- dependent plasminogen cleavage by 99%. These results suggest that activated Factor B, the central serine esterase of the APC, can serve as a plasminogen activator and raise the possibility that Factor B may play a role in initiation of fibrinolysis as well as in complement-dependent humoral and cellular mechanisms of immunity.
Presence Of Receptor For Activated Factor VIII On The Surface Of Released Platelets
Authors: Brodén K , Andersson L-O
Published: July 2018

In normal plasma Factor VIII activity is associated with a series of high molecular weight glycoprotein complexes also containing von Willebrand Factor related activities. To study the possible binding of various forms of Factor VIII to released platelets, a solution containing Factor VIII was mixed with a dilute suspension of platelets, which were released by addition of collagen. After 10 minutes of incubation the mixture was layered over 1.5 ml of 30% human serum albumin solution in a centrifuge tube and subjected to centrifugation at 7,000xg. Fractions were collected and analyzed for Factor VIII activity and phospholipid-related procoagulant activity. When purified Factor VUI/von Willebrand Factor complex was studied no significant association between the Factor VIII activity and the platelets were found. When purified Factor VUI/von Willebrand Factor complex was activated with 10-3 units/ml of thrombin and then tested, the main part of the Factor VIII activity became associated with the platelets. Even at very low platelet counts this binding was clearly detectable. The binding occurred both in the presence and in the absence of Ca2+. Thus released platelets bind thrombin-activated Factor VIII but not the Factor VUI/von Willebrand Factor complex. It is known that activation of Factor VIII by thrombin causes dissociation of the Factor VIII from the von Willebrand Factor part of the complex. The data obtained indicate that this dissociation is necessary in order to get the Factor VIII to bind to the platelet receptor. It may work as an amplification mechanism where the first traces of Thrombin formed upon initiation of coagulation dissociates Factor VIII from von Willebrand Factor, followed by binding to receptor on released platelets and formation of Factor X activator complex on the surface of the platelets.
Intrarenal Localisation Of Platelet Related Antigens, PF4, β-TG, Fibrin And Factor VIII In Patients With Glomerulonephritis
Authors: Duffus P , Parbtani A , Frampton G , Cameron J S
Published: July 2018

In vivo platelet activation is common in glomerular disease. A strone correlation between platelet serotonin depletion and the factors in the serum which affect platelet aggregation in vitro suggests that platelet activation predominantly occurs in the peripheral circulation. However, platelet activation could occur in the renal microvasculature resulting in the deposition of platelet related antigens at this site. We studied 88 biopsies by immunofluorescence microscopy for the presence of platelet membrane antigen(s), PF4, β-TG, fibrin and factor VIII. β-TG was not observed in any of the biopsies studied. Most biopsies (92%) showed positive endothelial fluorescence for factor VIII antigen, but were otherwise negative. Biopsies from patients with usually benign forms of glomerular disease (minimal change, focal and mesangial proliferative glomerulonephritis) were mostly negative; only 8% showing weakly positive fluorescence for platelet membrane antigens and PF4. Biopsies from patients with usually progressive forms of glomerulonephritis (MCGN, membranous nephropathy, PAN, SLE) were mostly positive (85%) for platelet membrane antigen(s) and PF4. A notable exception was biopsies from patients with FSGS; only two out of 11 biopsies (18%) were positive in this group of patients. The presence of platelet related antigens in glomerulonephritis suggests platelet activation and trapping at this site as well as in the peripheral circulation.
Evaluation Of Antithrombin III In The Prevention Of Intravascular Coagulation In The Rabbit
Authors: Triantaphyllopoulos D
Published: July 2018

In order to find out whether antithrombin III (AT III) alone in the absence of heparin is able to inhibit intravascular coagulation, 21 rabbits were infused under anesthesia with 0.19 - 1.74 mg/kg body weight/minute of rabbit brain tissue factor (TF) through the marginal vein of the ear for a maximum period of 1.5 hour and served as controls. Another group of 5 animals was injected with a bolus dose of human AT III, obtained by affinity chromatography on heparin-Sepharose, which was calculated to raise the concentration of the inhibitor by about 3 International units/ ml. This was followed by the infusion of TF at the same rates as in the controls. Blood samples were obtained through a catheter inserted in the carotid artery starting before the infusion of TF. Of the 21 control animals, 9 survived, 2 died in about 2 minutes and 10 between 15 and 30 minutes. Of the 5 animals which were preinfused with AT III only one survived (1.5 hour); the other 4 died about 15 minutes after the beginning of the infusion. No significant differences were found between control and experimental animals regarding the decline in the platelet count and in the concentration of fibrinogen, prothrombin, AT III, factors VIII, V, X and thrombin activity. In conclusion, preinjection of AT III alone, without heparin, into rabbits does not alleviate the manifestations of disseminated intravascular coagulation which are induced by the infusion of TF.
Quantitative Endotoxin Determination In Blood With A Chromogenic Substrate
Authors: Thomas L L M , Sturk A , Schaap M C , ten Cate H , ten Cate J W
Published: July 2018

A method, its optimalization and a solution to specific problems is described for the quantitative determination of endotoxins in blood. The method is based upon the endotoxin-dependent activation of a proenzyme, present in limulus ame- bocyte lysate. This activated enzyme is measured with the chromogenic substrate S2222. Possible inhibitors and activated coagulationfactors interfering in the assay are removed by dilution and boiling. The method is shown to be fast (2½ hr), sensitive and reproducible with a detection limit of 10 pg/ml.

The endotoxin assay was found to have a good correlation with results of blood cultures.

The detection threshold of the endotoxin assay compared with the number of circulating gramnegative organisms and CRD-measurements will also be shown.
Simple Chromogenic Peptide Substrate Assays For Determining Components Of The Plasma Kallikrein System
Authors: Gallimore M J , Friberger P
Published: July 2018

Over the past few years the introduction of chromogenic substrates has facilitated studies on components of the plasma defence systems. During this period it has become apparent that there is a need for simple, easy to perform, assays for measuring components of these systems. We have therefore developed simple assays for determining prekallikrein (PKK), kallikrein inhibition (KKI) and kailikrein-1ike activity (KK). These assays have a plasma dilution step, followed by repeat single volume pipettings of reagents. Both “rate” and “end-point” assays have been developed. For the PKK assay a new activator has been developed which contains Hageman factor and high mol. wt. kininogen. This overcomes the problems found by several groups with other activators when studying patients with low plasma levels of Hageman factor, PKK and high mol. wt. kininogen. For the KKI assay a stable preparation of human plasma KK has been produced. This assay detects mainly Cl'- esterase inhibitor. The assay for KK-like activity is required where activation of PKK has occured and spontaneous activity towards the substrate is found. Plasma samples from 25 normal donors were tested for PKK, KKI and KK. The PKK and KKI values were calculated from standard curves made with a standard plasma. When compared with PKK and C1INH antigen levels correlations of 0.84 and 0.85 were obtained. All of the plasmas had KK activities lower than 5% of the total PKK levels.

These assays should prove valuable in the rapid evaluation of the plasma KK system in patients.
Platelet And Coagulation Behaviour During Open Heart Surgery
Authors: Sturk A , Hoogendijk E M G , Schaap M C L , Sebel P S , Bovill J G , Beiboer J L , ten Cate J W
Published: July 2018

Studies were undertaken in 19 patients (16 m, 3 f) having open heart surgery. Parameters investigated were: hematocrit; whole blood platelet count; β-TG levels in plasma and platelets; aggregation by ADP, adrenalin and ristocetin; glycoprotein analysis of whole platelets; plasma levels factor II, X, plasminogen (plg), antithrombin III (AT III) and α2antiplasmin (ap). Samples were taken before (1) and 5 min after induction (2), five min after opening the sternum (3), at aortic canulation (4), 15 min after start of extracorporeal circulation (5) and at release of aortic crossclamp (6). Measurements in 5 and 6 were corrected for dilution. Platelet count was decreased 20-30% in samples 5 and 6. There was a 350% increase in plasma β-TG levels in sample 6, without a change in the circulating-platelet β-TG content. The increase therefore appears to be due to destruction or release of the non-circulating platelets. ADP-aggregation was normal in all samples, Adrenalin and ristocetin aggregation was absent in samples 5 and 6 due to the pump priming fluid (haemaccel). However, in sample 4 aggregation induced by ristocetin (1 mg/ml) was also absent. This could not be explained by the presence of haemaccel or heparin. Absence of GP IB (the possible ristocetin receptor) also could not be demonstrated. Plasma II, X, pig, AT III and ap did not change in sample 1-4. Factor II and X decreased 8-9% in sample 5 and 6, whereas plg, AT III and ap resp. decreased 12, 14 and 20%. Evidently activation of the coagulation starts with the extracorporal circulation.
Comparison Of The Thrombogenic Potential Of Two Membranes During Haemodialysis
Authors: Rajah S M , Crow M J , Donaldson D R , Sreeharan N , Davison A M
Published: July 2018

The thrombogenicity of two dialysis membranes was compared by observing the changes in platelet function, heparin sparing effect and scanning electron microscopy (SEM) findings following haemodialysis. Ten patients undergoing regular dialysis treatment using a Meltec Multipoint Kiil board dialyser of 1.5 sq.m, surface area, have been investigated. The two membranes studied were Cuprophane (C) and Polycarbonate (P). All patients entering the study had not received any antiplatelet therapy in the 14 day period prior and the heparin schedule remained unchanged for both membranes. The thrombogenic potential was assessed by chaig- es in platelet count, platelet adhesion, platelet aggregation with collagen, plasma B-Thromboglobulin (B-Tg) during dialysis, changes in heparin concentration post-dialysis and detailed examination of the membranes by SEM at the end of dialysis.

There was a significant fall in platelet count (p< 0.001) and platelet aggregation with collagen (p<0.05) with the C membrane, whereas the comparable values for the P membrane were not significant. Plasma B-Tg was very significantly raised with the C membrane (p<p.001) but to a lesser degree with the P membrane (p<0.05). Plasma heparin concentrations were significantly higher at the end of dialysis (p<0.05) with the P membrane compared to the C membrane, indicating a heparin sparing effect. SEM demonstrated consistently less platelet aggregates and fibrin deposition with the P membrane.

The results of this study indicate that the polycarbonate membrane has less thrombogenic potential (hence greater overall efficiency) than the Cuprophane membrane.
Need For Large (But Simple) Trials
Authors: Peto R , Yusuf S
Published: July 2018

Every year at least a million people are admitted to hospital with a diagnosis of suspected acute MI. Within a year about 15% are dead, some in hospital and seme after discharge. Reduction of 15% to (say) 12% would save same 30,000 lives per year, perhaps 10,000 of whom would be people in middle, rather than old, age. Such a reduction might well be conferred by certain of the treatments currently envisaged (e.g. limitation of infarct size by intravenous beta-blockade or by fibrinolytic treatment, or maintenance for several months following discharge on antiplatelet, anti-coagulant, beta-blocking or various other drugs), though it is much less plausible that large risk reductions (e.g. from 15% dead to 5% or 10% dead) could be conferred. Most trials are far too small to detect a reduction from 15% dead to 12% or 13% dead; for example, the mean size of the 36 randomised trials of beta-blockers or of fibrinolytic agents in the acute phase of (suspected) MI is 250 patients per trial. Such trials cannot reliably assess the sort of moderate reductions in mortality which it is medically realistic to hope for. This ideally requires not 250 per trial but more like 10,000 per trial. Some treatments (e.g. fibrinolytic agents, acute-phase i.v. beta-blockers, and post-discharge anti-platelet agents) which have failed to gain wide acceptance do seem to prevent death when all of the relevant trials are viewed together.
If such an overview of many trials is not accepted as sufficient evidence, the need for a few really large trials will be underlined. There is no need for expensive investigation of each patient in, large trials whose chief aim is to study mortality, and there may be more prospect of success in such trials if they are made unusually simple.

The point is not that all trials should be replaced by large, simple trials, but rather that unless a few large, simple trials are undertaken we shall not know which treatments prevent death.
The Use Of Peptide Fluorogenic Substrates In Cytochemistry
Authors: Smith R E
Published: July 2018

Cytochemical demonstration of proteases by fluorescent techniques is only a recent achievement. Localization of cellular proteases through the use of synthetic peptide substrates (as opposed to use of antigen-antibody in immuno-cytochemistry) depends upon the enzyme-substrate complex theory which assumes combination of enzyme with substrate in phase I and liberation of the enzyme and products in phase II. Determination of intracellular protease activity depends on liberation of a fluorophore attached to the peptide by an arylamide or ester bond. To localize the site of enzymatic hydrolysis of the substrate, the product must remain immobilized. Overcoming diffusion of reaction product is not a new problem in chromometric detection of enzymes, but it is enhanced with fluorometric detecting systems. Fluorescence microscopy allows for more precision in localizing reaction product, therefore diffusion is more noticeable. In flow cytometry, monodispersed cells are in a liquid transport media; thus diffusion of reaction product from a cell is unacceptable for it may be entrapped by another cell.

Although peptide derivatives of naphthylamines have been used in cytochemistry as chromogenic substrates for over 20 years, their fluorogenic potential was not useful until development of the concept of wavelength shifting. Naphthylamines fluoresce at short UV wavelengths, so the reaction product is difficult to quantitate above background interference. Furthermore, the product is soluble and easily washed out of cells or tissue. Coupling of 4-methoxy-2-naphthylamine to 5-nitrosalicylaldehyde provides a method of reaction product immobilization as well as extending the emission wavelength into the yellow-orange region of the spectrum. Nevertheless the technique has certain limitations, unavoidable in a two-step reaction product capture method. Recently, we synthesized peptide derivatives of several types of coumarins; some are proving to be useful fluorogens in the cellular localization of proteases.
Qualitative Defect In The Factor VIII/Von Willebrand Factor Protein (FVIII/VWF) In A Family With Hereditary Hemorrhagic Telangiectasia (HHT)
Authors: Conlon C , Weinger R , Moake J , Rudy C
Published: July 2018

A 63 year old man with HHT has a lifelong history of recurrent epistaxis and a 6 year history of gastrointestinal bleeding which required frequent transfusions. He had a normal prothrombin time, activated partial thromboplastin time, thrombin time, template bleeding time, platelet count and platelet aggregation studies. Factor VIII-coagulant activity, factor VIII-VWF and factor VIII-VWF related antigen levels were elevated. On two-dimensional crossed immunoelectrophoresis (2D-CIE) his plasma FVIII-VWF antigen had abnormal, anodal mobility on repeated testing of different plasma samples. Patient platelet-rich plasma (PRP) did not agglutinate in response to ristocetin concentrations lower than required to agglutinate normal PRP, as is the case in type IIB von Willebrand’s disease. However, binding of patient plasma FVIII-VWF antigen to normal formalin-fixed platelets occurred at lower ristocetin concentrations than required to induce binding of normal plasma FVIII-VWF antigen. Similar clinical and laboratory features were present in his 61 year-old clinically affected sister, but not in his unaffected brother. Because of the abnormal 2D-CIE, 10 units of normal cryoprecipitate were infused. Fifteen minutes later, 2D-CIE of patient plasma FVIII-VWF antigen was normal; by 8 hours mobility was abnormally rapid again. During the next weeks, his RBC transfusion requirement was not affected by infusions of cryoprecipitate (6 units) three times weekly, or by daily e-aminocaproic acid (24 grams). Because glucocorticoids suppress endothelial cell synthesis of the anti-aggregation substance, PGI2, he was given a trial of daily prednisone (20 mg.). Concurrently, his RBC transfusion requirement decreased by ̴75%. HHT (with abnormal endothelial cell proliferation) in this family is associated with a qualitative defect of plasma FVIII/VWF protein and, possibly, with prednisone-responsive excessive local production of PGI2.
Acquired F VIII Inhibitor In An Identical Subhemophilic Twin
Authors: ten Cate J W , Sturk A , Breederveld C
Published: July 2018

An identical (94,3% certainty) twin developed a factor VIII inhibitor, almost simultaneously, which may imply a genetic predisposition. Both patients were high responders (18 an 22 Bethesda U). The initial FVIII;C plasma levels gradually decreased from 0.24 U to 0.08 U. Upon transfusion of 2500 U human FVIII concentrate 75% was inactivated immediately and the remaining factor VIII;C activity had a T½ of 2 h. In order to investigate the T½ of their own FVIII, plasmaphoresis was performed in one of them and 500 ml plasma was cryo- precipitated as the first step for factor VIII purification. Surprisingly, only 10% of the FVIII;C present in plasma was recovered in the cryoprecipitate. FVIII was purified by Sepharose 6B chromatography. Inhibitor activity eluted in later fractions seperate from FVIII. The void volume fractions were pooled and were negative for FVIII inhibitor activity. Purified FVIII was radiolabeled (125I-lactoperoxidase technique) . CIg followed by autoradiogrphy and PAGE of the IFVIII, gave normal results. 12 u Cl 125I FVIII was injected i.v. and revealed a biphasic normal half life: T½ of the first phase was 2.0 h, of the 2nd phase 19.2 h. This may imply that this patient developed antibodies selectively directed against FVIII neoantigens present in the FVIII concentrates (T½ h:) . However, i.v. administration of DDAVP resulted in a decreasing FVIII inhibitor titer presumably due to complex formation of released endogenous FVIII the inhibitor. It is therefore concluded that 125I VIII formes complexes with the inhibitor and that these complexes are slowly cleared from the circulation.
Prediction Of Stability Of Thromboplastin
Authors: Stevenson K J
Published: July 2018

A new, rapid degradation method for predicting the stability of lyophilised extracts of tissue thromboplastin by accelerated oxygenation is described. This requires less time to complete and is more relevant to tissue thromboplastin age degradation than heat-accelerated methods previously advocated. Lipid oxidation is an important contributor to age-related change of thromboplastins. Although high teinperatures employed in heat degradation may increase oxidative processes in thromboplastin lipids, the protein component essential for procoagulant activity is also denatured.

The practical application of the rapid oxygenation method as a technique for accelerated degradation of tissue thromboplastin reference preparations is described.
The Effect Of Different Activators And Thromboplastins On A Chromdgenic Aptt
Authors: Brandt J T , Triplett D A , Schaeffer J
Published: July 2018

Four different activator/thromboplastin reagents (silica/rabbit brain cephalin, kaolin/ simian brain cephalin, ellagic acid/rabbit brain cephalin, and ellagic acid/bovine brain cephalin) were evaluated for use in a chromogenic activated partial thromboplastin time (APTT). Parallel determinations using the same reagents in a clotting APTT were made. Compared to the clotting APTT, the chromogenic APTT using ellagic acid activation showed much greater sensitivity to both in vivo and in vitro heparin. Silica activation in the chromogenic assay showed decreased sensitivity to in vivo and in vitro heparin compared to the clotting assay. The kaolin/simian brain cephalin reagent was relatively insensitive in both clotting and chromogenic assays. A significant difference between in vivo and in vitro heparin was noted with all of the reagents in both clotting and chromogenic assays.

The chromogenic assays were more sensitive to the effect of vitamin K deficiency and coumadin administration than the corresponding clotting assays. Relative to the effect on the clotting APTT, the chromogenic assays were more sensitive to coumadin and vitamin K deficiency than to in vivo heparin.

The results indicate that the chromogenic APTT is not equivalent to the clotting APTT. Distinct differences in sensitivity to heparin and coumadin exist between the two assay systems. There is a marked difference in response to different reagent systems in the chromogenic assay. These various effects need to be considered when designing chromogenic assays.
The Effects Of An Anticoagulant Phospholipase A2 On The Procoagulant Activity Of Phospholipids And Platelets
Authors: Cox A C
Published: July 2018

Although phospholipids readily substitute for platelets in many in vitro blood coagulation tests, their participation in normal platelet procoagulant activity is uncertain. Phospholipase A2 (PLA2) from Naja nigricollis nigricollis venom, a known anticoagulant, blocked the enhancement of the rate of prothrombin conversion to thrombin by both phospholipids and platelets. The rate of inhibition of the phospholipid procoagulant activity by PLA2 was reduced by indomethacin, an inhibitor of PLA2. At concentrations of PLA2 which inhibited phospholipid procoagulant activity, conversion of purified prothrombin by factors Xa and Va without phospholipid was unaffected.

Unactivated, washed platelets combined with coagulation factors II, Va and Xa initially produce thrombin at a slow rate before the platelets become activated but after this lag period the rate of thrombin generation increases. PLA2 added at the same time as the coagulation factors increased the lag period and decreased the rate of thrombin generation during and after the lag. Preincubation of platelets with PLA2 further decreased only the lag rate and this inhibition was partially blocked by 300 uM indomethacin. At a concentration of about one ng/ml, PLA2 reduced the post-lag thrombin generation rate of 3 × 10-9 platelets/ml in half but had no effect on platelet aggregation induced by thrombin, ADP or collagen.

These results combined with the known specificity of PLA2 support the theory that phospholipids are involved in platelet procoagulant activity. Furthermore, the ineffectiveness of preincubating PLA2 with platelets on the post-lag procoagulant activity suggests that the phospholipids involved in this post-activation process become accessible during the lag period.
The Effects Of Platelets, Fibrinogen And Factor XIII In ‘Resonance-Thrombography’ (RTG)
Authors: Husar D , Hartert H
Published: July 2018

The method of ‘Resonance-Thrombography’ was invented by H. Hartert in 1970 and is now introduced as a screening test for various bleeding disorders. The RTG-curve mainly depends on the number of platelets as well as platelet-function, amount of fibrinogen and factor XIII.

A successful theoretical approach to describe this depen- dancy is given by means of a one-dimensional, linearized model of apparatus and coagulation process. The experimental results and the simulated curves agree within the experimental errors. The model further provides the possibility to derive time-constants and cross-linking-factors for the fibrinogen-polymerisation and the interaction of platelets with fibrin.
Binding Of Thrombin By Plasma Inhibitors
Authors: Penner J A , Hassouna H I
Published: July 2018

Our study was undertaken with the purpose of clarifying the role of plasma inhibitors other than antithrombin III (ATIII) in binding thrombin in the presence and absence of heparin. In the clotting system, several antiproteinases are involved in neutralizing active factors. The capacity to destroy thrombin is attributed to ATIII whose concentration in plasma is ten times less than α1 antitrypsin (α1A) . If heparin is added to plasma, ATIII becomes an immediate inhibitor of thrombin. Several studies have indicated that a modest depression in ATIII activity leads to thrombosis, inferring that other plasma antiproteinases play a minor role. Experimental approach was as follows: Defibrinated platelet poor citrated human plasma was depleted of ATIII and α1 by means of monospecific insolubilized antibodies. Purified thrombin, labelled by Chloramine T incorporation of 125I in NaOH, had a specific activity of 1.3 × 105 CPM/μg. Total antiproteinase thrombin binding capacity of defibrinated plasma was estimated by incubating 4 × 10-33 μM thrombin with plasma (100 μl) for 2 hours and 24 hours in the absence of heparin and for 5 minutes in the presence of heparin (20 units). Free from bound *thrombin was separated by gel filtration on Sephacryl S200 and chromatograms monitored on gamma analyzer. Following the same protocol, ATIII deficient plasma was used to evaluate thrombin binding capacity not attributable to ATIII, and role of ATIII in the absence of α1A was assessed in α1A depleted plasma. As a result, ATIII binding capacity was found to be 62 μg *thrombin/100 μl of normal and α1A deficient plasma, in the presence and absence of heparin, eluting at a volume corresponding to 9 × 104 daltons. Binding capacity of other plasma inhibitors in ATIII deficient plasma amounted to 24 μg *thrombin in the absence of heparin and 7 μg in its presence. This confirms the role of ATIII as the major antithrombin, heparin cofactor. In its absence inactivation of thrombin is reduced by 61%.
Effect Of T-2 Mycotoxin On The Coagulation Mechanism
Authors: Gentry P A , Cooper M L
Published: July 2018

Corn and wheat stored under low temperature conditions can become contaminated by the mold Fusarium Trincinctum. Animals affected with com toxicosis develop symptoms of epistaxis, anorexia and hemorrhagic lesions of the intestinal and urinary tracts. Controversy exists concerning which of the several metabolites produced by the mold is responsible for the hemorrhagic syndrome. Studies from this laboratory have shown that one of the metabolites produced by the fungus, T-2 toxin (4,15- diacetoxy-8-[3 methylbutryloxy]-12, 13-epoxy-Δ9-tricothecene-3-ol), can induce a prolongation of the plasma clotting time when determined by either the activated partial thromboplastin time assay or the one stage prothrombin time assay. A single intravenous administration of T-2 toxin to either rabbits or calves, at a dosage of 0.50 and 0.25 mg per kg body weight respectively, results in a decrease in the plasma activity of Factors VII, IX, X and XI of 40-50 percent of pretreatment values. The maximal decrease in activity is observed 24 hrs after toxin administration and the activity remains depressed for the next 48 hrs before gradually returning to initial values. Plasma fibrinogen concentration decreases from 498 gm/1 to 395 gm/1 by 24 hours and to a minimum of 351 gm/1 by 54 hrs before showing a partial recovery. Platelet counts remain unchanged. Preliminary data indicate that the T-2 toxin does not act directly as a vitamin K antagonist in producing the alteration in procoagulant activity.
Intensity Of Therapeutic Range And Mean Anticoagulant Dosage Prescribed: An International Study
Authors: Poller L , Taberner D A
Published: July 2018

It has been suggested that anticoagulant dosage requirements vary in different parts of the world. An international survey on anticoagulant dosage involving over 500 laboratories was performed employing a questionnaire on the mean daily anticoagulant dosage of 20 stabilised patients. Replies received show a considerable variation in dosage between countries.

In order to further investigate the influence of the intensity of the therapeutic range on local dosage differences, participants were asked to assess a lyophilised human coumarin plasma sample. Most agreed that the plasma was adequately anticoagulated. However, laboratories finding the plasma overdosed were prescribing a lower mean anticoagulant dosage than those laboratories finding the plasma underdosed. This suggests that some of the differences in dosage between laboratories may be accounted for by the varying intensities of the therapeutic ranges used in the different countries.
Competitive Enzyme Immunoassay For Fibrinogen And Fibrinogen/Fibrin Degradation Products
Authors: Ho T , Gray A
Published: July 2018

A competitive enzyme immunoassay for fibrinogen and fibrinogen/fibrin degradation products (FDP) is described, in which a peroxidase - fibrinogen conjugate competes with fibrinogen or FDP for binding to a fibrinogen antibody attached to polystyrene solid phase. After this competition reaction is completed, unbound fibrinogen or FDP is removed by washing the polystyrene-antibody complex with water. The extent of the conjugate binding to antibody is observed by adding a chromogenic substrate for the peroxidase reaction. Peroxidase activity measured spectrophoto- metrically at 492 nm is inversely proportional to the fibrinogen or FDP concentration in the serum sample. This enzyme immunoassay requires only 50 ul of human serum per assay and can be performed in as little as 45 minutes. Using this method, serum collected from normal healthy volunteers give a mean FDP level of 1.8±0.7 ug/ml (n=24). With a properly anticoagulated plasma, the assay can be used to determine immunological fibrinogen level in plasma sample. This enzyme immunoassay is unaffected by the presence of rheumatoid factors in some sera, which has been reported as a source of error in latex agglutination technique. The assay has the added advantage of measuring directly the concentration of FDP in serum, without making serial dilution of the sample.
A Quantitative Normal Fibrin Plate Assay
Authors: Devine E M
Published: July 2018

Astrup and co-workers introduced the normal fibrin plate assay method for assaying plasminogen activators in 1952. Over the years, this technique has been used as a semiplasminogen activators such as urokinase. Our laboratory designed experiments to investigate and establish parameters for the fibrin plate assay which would give a maximum and reproducible response to quantities of urokinase. The parameters investigated included molarity (ionic strength) and pH of the buffers used for both the fibrin clot and the unknown sample. To establish these conditions, a series of experiments were performed involving parallel line assays. With this procedure, the response to each parameter studied was compared at several different levels of activity. Statistical comparisons were made to determine how changes in the measured parameters would influence the sensitivity and precision of the assay. The outcome of these studies led to the development of a routine normal fibrin plate assay for urokinase using a sample pad and a standard curve with an activity range of 0.1 to 0.8 IU. These conditions yielded a linear response on a semilogarithmic scale. When assaying urokinase, the results of the conventional fibrin plate assay (one level unknown vs. four levels standard) and the dose response-parallel line-fibrin plate assay (four levels unknown vs. four levels standard) are equivalent. The coefficient of variation for both assays was 2 to 5 percent.
Development Of A Fully Automated Functional Fibrinogen Assay
Authors: Siefring G E , Wehrly J A
Published: July 2018

A fully automated method for the determination of functional fibrinogen has been developed for the Du Pont ‘aca’. The basis of the assay is the measurement of turbidity formation upon the addition of thrombin to the test plasma. The method can measure fibrinogen in the plasma of patients on heparin therapy and provides a stat result in eight minutes.

The assay of fibrinogen is accomplished in a two-stage sequence. First, clot enhancing agents and a heparin neutralizing compound are added. Thrombin is added in the second step to catalyze fibrin formation, the rate of turbidity change being proportional to the fibrinogen concentration. Optimization of the assay under ‘aca’ conditions was accomplished through the use of multivariate response surface analysis. The analytical performance of the method was judged to be acceptable based on precision, interference, and correlation studies. The range of the method is 40-1000 mg/dl fibrinogen. This ‘aca’ method represents the first fully automated determination of functional fibrinogen.
Fibrinogen In Liver Cirrhosis. Lack Of Correlation Between Soluble Fibrin Monomer Complexes And Fibrinopeptide A Levels
Authors: Coccheri S , Mannucci P M , Palareti G , Cattaneo M , Poggi M , Vigand S
Published: July 2018

In this collaborative study we have investigated the coexistence of two different markers of fibrinogen to fibrin conversion in 19 patients with liver cirrhosis, confirmed with liver biopsy, and classified according to standardized criteria into 11 moderate and 8 severe cases. Sepharose gel filtration of plasma allowed separation of fibrinogen-like material (FLM) into high m.w. (HMWF), peak, and low m.w. (LMWF) fractions, quantitated as percent (w/w) of the total FLM eluted. FPA was assayed radioimmunologically.

HMWF was markedly increased in the whole group of cirrhotics (av ± SD 10.4% ± 2.77) , and in both moderate and severe patients, vs controls (6.9% ± 1.39; n=16; p < 0.01 for each comparison). LMWF was moderately increased in patients (6.8% ± l.84) vs controls (5.3% ± 1.35; p < 0.05).

FPA levels (controls: median (m) ng/ml 2.2; m confidence limits (mcl) 1.4-2.5; range (r) < 0.6 to 4.1) showed an increase in cirrhotics (m 2.8; mcl 2.64-3.35, r<0.6 to 94) but the difference was not statistically significant using a non-parametric test. However, in 6 out of 19 patients FPA values exceeded av ± 2 SD of controls (av ± SD 2.05 ± 1.15). No significant correlations were found between FPA and HMWF or LMWF levels.

The lack of correlation between FPA and HMWF although possibly related to half-life differences, suggests that, besides thrombin effect, other mechanisms for excess formation and/or accumulation of HMWF, as dysproteinaemia, dys- fibrinogenaemia, or impaired clearance, should be considered.
Functional And Biochemical Studies On A Case Of Dysfibrinogenemia (Fibrinogen Genova)
Authors: Hassan H J , Orlando M , Mori P G , Tonini G , Casalbore P , Sarti A , Boeri E , Tentori L
Published: July 2018

Fibrinogen Genova was recognized in an Italian family. The propositus had no bleeding tendency and diagnosis was performed on the basis of prolonged prothrombin, thrombin and reptilase times of plasma, associated with normal fibrinogen levels. The purified fibrinogen showed an inhibitory effect on the clotting times of the normal one.

Functional studies performed on purified fibrinogen revealed that the clottability was reduced (74%, normal 88-94%) and the thrombin polymerization curve was abnormal due to a delayed fibrin monomers aggregation. The fibrino- peptides release was normal.

Analysis of plasmic derivatives carried out by both SDS gel electrophoresis (5 and 9%) and immunological methods showed some differences compared with normal fibrinogen. The fibrinogen chains were studied on isoelectric focusing slabs and SDS gel electrophoresis. Preliminary results did not reveal any difference with normal.
A Comparison Between Thrombotest And Factor-X Amidolytic Activity In Stable And Non-Stable Anticoagulated Patients
Authors: van Wijk E M , Kahlé L H , Jeletich A , ten Cate J W
Published: July 2018

We tested a mechanized amidolytic factor-X assay in 2222 patients on long term anticoagulant therapy. A good correlation was found between this assay and the routinely performed Thrombotest (r= 0.78). With a therapeutic range between 150 and 300 units/L of factor-X amidolytic activity we obtained the same information about the state of anticoagulation in 81 % of these 2222 patients. Factor-X activity was assayed in 32 stable anticoagulated patients together with Thrombotest on at least three subsequent occasions in each patient. A fairly constant Thrombotest-factor X ratio was observed.(mean 0.38±0.06) Changes in Thrombotest and factor-X amidolytic activity ran consistently parallel in a group of 20 unstable anticoagulated patients.(mean ratio 0.45±0.08) Changes in Thrombotest were occasionally more pronounced than changes in factor-X activity in some patients, which could be due to the high turnover of factor VII. Relative very high or low factor-VII levels as compared to the other vitamin-K dependent factors disturbed the balance between Thrombotest and factor X. The therapeutic consequence in these unbalanced states is, that the prescribed dosage scheme of the anti-vitamin K drug could depend on the very moment of blood sampling. As a result of the high turnover of factor VII, bloodsampling one day earlier or later would then result in another dosage scheme of the drug. Therefore it might be concluded that oral anticoagulant therapy, guided by a factor-X assay could result in more stable long term treatment. Large prospective comparative trials are required to support this hypothesis.
Biological Characteristics Op Antithrombin III In An Antithrombin III Deficient Family
Authors: Kondo S , Matsuo T , Ohoki Y , Matsuo O
Published: July 2018

In the familial AT III deficiency of a Japanese family, the propositus (a-39-yr old female) and her mother had episodes of recurrent thrombosis and their AT III levels as measured immunologically and biologically were below the normal value. In the plasma of her brother, the AT III concentration as measured immunologically was half of the normal value, but his biological antithrombin activity was within the normal range. The progressive antithrombin activity and antifactor Xa activity of plasma samples in this familial AT III deficiency were within the normal range. Measurements of the rate of thrombin neutralization activity revealed that the brother’s plasma was in the normal range, but the plasma of the propositus and of her mother showed rates of thrombin neutralization activity which were somewhat below the normal value. The rate of thrombin neutralization activity per mg protein of AT III was highest in the plasma of the brother, and became slower in the mother, propositus, and pooled normal plasma in that order. In the plasma of this familial AT III deficiency, the rate of Xa neutralization activity was much slower than the normal value. It is postulated that since the antithrombin of the brother of the propositus was found to react as normal in the neutralization of thrombin, he does not have episodes of thrombosis. Such characteristic hyperfunction of antithrombin in the plasma of the brother may be due to some molecular abnormality of AT III within this hereditary deficient family.
On The Effective Heparin Therapy With No Complications
Authors: Oguma Y , Takei H , Seya T , Murakoshi T , Yamauchi M , Nagata H , Hasegawa H
Published: July 2018

In order to establish the effective heparin(HP) therapy without any complicated episodes, we investigated the changes of plasma HP concentrations (COATEST HP, KABI), APTT, FPA, AT III activity(AT III F), AT III immunology(AT III I), and PF 4 in 40 patients receiving HP treatment (15 cases in DIC, 25 cases in the other disease). There was a correlation between HP concentrations and APTT. However, the regression lines varied significantly in each case, especially between DIC group* and non-DIC group*(p<0.005). So, it was suggested that single estimation of HP concentration or APTT might not give a correct information and both estimations were required for appropriate HP therapy. In the patients with complete remissions obtained by HP administration, FPA improved most initially and normalized continuously thereafter in the range of 0.2-1.2 IU/ml of HP concentration and 45-250 sec. of APTT and no hemorrhagic incidents occurred. Considering FPA is the most sensitive indicator of thrombin action on fibrinogen, we are convinced that this range is the effective therapeutic range with no complications. AT III F and AT III I showed a good correlation (r: 0.827, p<0.01) but the ratio of AT III F/AT III I was prominently depressed in acute decompensated DIC. In most cases, both AT III levels were decreased reflecting the pathologic consumption. The HP treatment for these cases led further decrease of AT III for a couple of days and returned afterwards to normal levels in HP effective cases, while decreased more in HP ineffective cases. The minimum AT III levels indispensable for HP therapy were also considered 40% of normal values. In thrombotic diseases, PF 4 was elevated with a good correlation to βTG, except in patients with extremely low platelet counts. But the changes of PF 4 was not always coincident with FPA changes. When HP was administered to normal subjects, elevated PF 4 levels were found while βTG remained normal, suggesting the different release mechanism of PF 4 and βTG.
Prevention Of Deep Vein Thrombosis In Medical Patients By Low Dose Subcutaneous Heparin
Authors: Belch J J F , Lowe G D O , Ward A G , Forbes C D , Prentice C R M
Published: July 2018

In recent years it has been repeatedly shown that low-dose subcutaneous heparin reduces the incidence of deep vein thrombosis (D.V.T.) after major general surgery. By comparison, the prevention of thrombosis in medical patients has been little studied. A randomised trial was undertaken in one hundred patients with heart failure and/or chest infection to determine whether low-dose subcutaneous heparin reduced the frequency of D.V.T. in the legs. Heparin (5000 units 8 hourly), started within 12 hours of admission to hospital and continued until the patient was fully mobile, significantly reduced the frequency of D.V.T. diagnosed by the 125I- fibrinogen scan technique, from 26% to 4% (p<0.01). Heparin did not cause bleeding problems except for a 20% incidence of injection site bruising. We therefore recommend prophylaxis with low dose subcutaneous heparin in patients with heart failure or chest infection who require more than 3 days’ bed rest.
Fibrinogen Binding Is Independent of an Increase in Intracellular Calcium Concentration in Thrombin Degranulated Platelets
Authors: Pulcinelli Fabio M , Daniel James L , Riondino Silvia , Gazzaniga Pier Paolo , Salganicoff Leon
Published: July 2018

In a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C(PLC) activation and calcium mobilization in TDP activated by ADP, epinephrine and U46619.

ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.
Estrogens Reduce Plasma Histidine-rich Glycoprotein (HRG) Levels in a Dose-dependent Way
Authors: Hennis B C , Boomsma D I , Fijnvandraat K , Leuven J A Gevers , Peters M , Kluft C
Published: July 2018

Plasma levels of histidine-rich glycoprotein (HRG) were investigated in three groups of women receiving a different dose of estrogens. First, the effect of low-dose estrogen was studied in a group of 83 postmenopausal women who were treated with 0.625 mg conjugated estrogens (CE). No significant change from baseline levels was found at the end of cycle 3 and cycle 13. Secondly, in 15 mothers and 23 daughters using oral contraceptives (OC) containing 30-50 fig ethinyl estradiol (EE) daily the mean HRG level was 14% and 24% lower than in a group of 144 mothers and 134 daughters not taking oral contraceptives, respectively (p < 0.05). Finally, in 11 excessively tall prepuberal girls who received 300 μg EE daily to reduce their final height the mean plasma HRG levels were decreased by 68% (p < 0.005). The effect of proges- togens administered during low-dose and high-dose estrogen therapy appeared to be minor.

The results from these three studies indicate that estrogens reduce plasma HRG levels in a dose-dependent way.