bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotonin Release from Filtered and Unfiltered Human Platelets Induced by Antigen/Antibody Complexes of Different Valencies
Authors: Kazatchkine M. , Caen J. , Johnson Anthea H. , Mowbray J.F.
Published: May 2019

In order to study the nature of receptors for immune complexes (IC) on the surface of platelets, the release of radiolabelled serotonin produced by exposure of platelets to IC of known composition was studied. Free plasma components might alter this reaction, so platelets were used both in platelet rich plasma (PRP) and after gel filtration. As it has been thought that the aggregation of the immunoglobulin molecules through the antigen was neccessary for the release reaction, we have studied complexes made with antigens of different valencies. The antigen was albumen substituted with different numbers of dinitrophenol (DNP) groups, and the antibody rabbit-anti DNP. Polyvalent antigen complexes were also made from bovine serum albumen (BSA) and rabbit anti-BSA antibody. Soluble and insoluble IC made in a variety of ratios with polyvalent antigen induced release both in PRP and in gel filtered platelets. Soluble complexes in antigen excess were the most effective. Complexes of monovalent antigen prepared, using two methods of monovalent substitution, were also capable of inducing release in PRP and in gel filtered platelets, although the complex could not exist in an aggregated form. Release was less than that produced by polyvalent complex, but could be increased by the addition of CI.

These results show that, at least with monovalent IC, the simultaneous involvement of more than one receptor is not an absolute requirement for platelet release. These receptors for monovalent complexes could be different from the Fc receptors for aggregated immunoglobulins or polyvalent complexes.
Treatment of Congenital Factor VII Deficiency with a New Concentrate
Authors: Mariani G. , Man-nucci P.M. , Mazzucconi G. , Ruggeri Z.M.
Published: May 2019

A commercial factor VII concentrate, made from ACD plasma by a process involving successive adsorptions of cryoprecipitate supernatant on DEAE Sephadex and of the resulting supernatant on A1(0H)3 was administered to 10 patients with severe factor VII deficiency.5 patients received only one dose for treatment of single bleeding episodes, the remaining 5 were given multiple doses (47) for spontaneous hemorrhages or for the prevention of surgical bleeding. The factor VII recovery in vivo ranged from 43 to 126% (average 88%) of the assayed in vitro activity of the concentrate. A dose of 0.5 factor VII u/Kg b.w. was found to produce a 1% rise of plasma factor VII level. The mean half-life of injected factor VII as assessed in 7 kinetic studies was 205 min (range 168-234). Spontaneous bleeding was easily controlled by the concentrate and major surgical procedures (two tonsillectomies) could be performed without complications. One patient developed HBsAg positive hepatitis, but otherwise no serious side effects were observed. Factor VII concentrate reduces the risk of precipitating circulatory overload associated with the use of plasma and avoids the unnecessary rise of factor II, IX and X which follows prothrombin complex concentrates while still achieving hemostatic factor VII levels.
Prevention by Phospholipase C of Pulmonary Insufficiency Syndrome Induced by Tissue Thromboplastin in Rats
Authors: Giercksky K-E
Published: May 2019

Purified phospholipase C (PLC) is a potent inactivator of tissue thromboplastin in vitro (1.2). Rats injected with a lethal dose of purified human tissue thromboplastin (3) survived when given PLC i.v. before the thromboplastin injection (4.5). PLC i.v. also led to a striking reduction 125I-fibrin and 51Cr-platelets in the lungs when given just before a sublethal infusion of thromboplastin (5). Rat adipose tissue was homogenized and centrifuged to give 3 fractions, of which one had a marked procoagulant, tissue thromboblastin-like activity. Infusion of this fraction led to an accumulation of 125I-fibrin and 51Cr-platelets similar to that following infusion of tissue thromboplastin. LD50 for purified PLC in rats have been determined (6). Doses smaller than 10% of LD50 protected rats against otherwise lethal doses of the procoagulant from adipose tissue and reduced the accumulation of fibrin and platelets in the lungs to nearly control levels.PLC does not alter the primary bleeding time, platelet half-life or thrombin-induced platelet aggregation.1. Otnasss et al E.J.B. 1972, 2. Bjørklid et al TDH 1973, 3. Bjørklid et al BBRC 1973, 4. Giercksky et al S.J.H. 1976, 5. Giercksky & Bjørklid, S.J.H. 1976, 6. Otnæss et al S.J.C. lab. 1976.
Comparative Hematology: Studies on Class Aves, Domestic Turkey, Meleagris Gallopavo
Authors: Lewis Jessica H. , Hasiba Ute , Spero Joel
Published: May 2019

This study compared turkey and human blood in various coagulation and cellular parameters. Turkey blood clotted slowly (± 60 minutes) in either glass or siliconized tubes and the formed clots retracted very slightly or not at all. Prothrombin times, using mammalian brain, were 12-14 seconds for human plasma and over 60 seconds for turkey plasma. On the other hand, turkey brain clotted turkey plasma in 12 to 14 seconds and human plasma in over a minute. Russell Viper Venom clotted both plasma in 17 to 22 seconds. The activated partial thromboplastin time on turkey blood was over two minutes. Bovine thrombin clotted turkey plasma in about 30 seconds compared to 15 for human. Turkey clotting times with human thrombin or reptilase were very much longer than human. Thromboplastin generation tests with all human components gave substrate clotting times of 8.6 to 9.1 seconds; with all turkey components they were greater than 40 seconds. Turkey fibrinogen averaged 450mg/dl. Assayed in systems used for human coagulation factors, turkey factor XIII was high normal, factor VIII low normal (0.48U/ml), factors II and V present in traces and the others, VII, X, IX, and XII, absent or not measurable. Turkey thrombocytes were oval, nucleated cells which aggregated during clotting but not with adenosine diphosphate, mammalian or turkey collagen or ristocetin.TEM showed thrombocytes to contain microtubules, an open canalicular system, and a few mitochondria as well as a large nucleus. Hemostasis in turkeys appears dependent upon an extrinsic pathway in which tissue factor from an injured area initiates thrombin formation, which, in turn, causes thrombocyteaggregation and fibrin formation.
A Comparison of the Anticoagulant Actions of Commercially Available Contrast Media (CM)
Authors: Moneada Rogelio , Fareed Jawed , Messmore Harry , Demos Terrence
Published: May 2019

Previous studies have reported on the anticoagulant effect of commercially available contrast media used in diagnostic radiology. The purpose of this study is to compare the anticoagulant actions of these agents in vitro. Eight commercially available contrast medias were supplemented to citrated human plasma (CNP) in 1:10, 1:20 and 1:50 proportions; isomolar sucrose, glucose, sodium chloride, and saline supplemented CNP were used as controls. Prothrombin time (FT) , partial thromboplastin time (PTT), thrombin time (TT), antithrombin-III, plasminogenplasnun and factor assays were performed at 0 time, 30 minutes and 2 hours after incubation at 37°C. No significant alteration of the coagulation parameters were observed at 1:50 dilution, however at 1:10 and 1:20 dilution, most contrast media produced aberration of clotting parameters. The antithrombin potency of these contrast media at a 1:10 dilution ranged from 0.3-1.3 u/ml heparin. In addition, this antithrombin activity was synergistic with heparin. The antithrombin activity of these agents was not neutralized by protamine sulfate, polybrene or toluidine blue in the amounts which neutralized 1 u/ml heparin. Analysis of various clotting factors revealed that factors II, V, VII and XII were not affected by contrast media. Factors VIII and IX were depressed significantly. These changes were mainly dependent on the concentration of meglumine in the contrast media. Similar studies on the blood obtained from patients infused with contrast media for diagnostic purposes are in progress in our laboratory.
The Passovoy Defect: Further Case Reports and Preliminary Characterization
Authors: Hougie C. , Brown J.E. , Lakin-Thomas P. , McPherson R.A. , Aronson L. , Baugh R.F.
Published: May 2019

Since the first report of a patient with a deficiency of the Passovoy factor (Lancet 11:220, 1975) we have encountered 10 other cases from six kindred. The patients have a small but significant prolongation of the activated partial thromboplastin time which is corrected by normal plasma. They have normal levels of all the hitherto reported clotting factors, including Fletcher and Fitzgerald, no evidence of an inhibitor, and no mutual correction. The condition is transmitted as an autosomal dominant. Most of the patients have had a moderate to mild bleeding diathesis, thus the proband in the original kindred recently bled profusely into the neck three days following the removal of a small cervical node and was given 6 units of blood to control the bleeding. Attempts to characterize the factor have been hampered by the relative insensitivity of the assay method; however a fraction has been found which shortens the clotting time of Passovoy deficient plasma, but not plasmas deficient in other clotting factors; the biochemical properties of this factor are currently under investigation.

This work was supported by USPHS research grant HL 19272.
Pharmacokinetics of Human Factor IX in a dog with Severe Hemophilia B.
Authors: Gentry P.A. , Thompson A.R. , Forrey A.W.
Published: May 2019

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.

Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.

Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.
Detection of Antithrombin III /AT-III/ Complexes in “Hypercoagulable” and Hyper-Fibrinolytic States
Authors: Sas G. , Köves Á , Pető I.
Published: May 2019

As it was previously described, the complex of thrombin-AT-III end “free” /uncomplexed/AT-III can be discerned by a modified crossedimmunoelectro -phoresis method /heparin in agarose/. Further investigations with the same technique showed that activated factor X/Xa/ and plasmin also form complexes with purified AT-III which, Just as thrombin-AT-III complex, migrate slower, than “free” AT-III. On the basis of these observations we assumed a significant increase of the quantity of these AT-III fractions in various “hypercoagulable” and hyperfibrinolytic states, mainly in DIC.

Only about one third of patients with chronic DIC displayed significant increase of AT-III complexes in their plasmas. In a few patients with deep venous thromboses a similar increase of complexed AT-III was observed. In cirrhotic patients with low prothrombin level the administered PPSB concentrate brought about a mild DIC with a striking decrease of “free” AT-III concentra -tion but there was no increase of the complexes. Streptokinase therapy did not increase the quantity of the slow moving fractions of AT-III at all, but the concentration of “free” AT-III decreased significantly.

A fast in vivo clearing mechanism of the AT-III complexes can be postulated which could account for the absence of a significant increase of AT-III complexes while the “free” AT-III concentration decreases.
Venous Thromboembolism: A Comparison of Treatment with Intermittent Constant Dosage Heparin and Continuous Infusion with Laboratory Control
Authors: Mant M.J. , O’Brien B. , Grace M.G.
Published: May 2019

We are conducting a prospective randomized trial comparing the treatment of venous thromboembolism for 10 days with constant dosage intermittent (I) I.V. heparin 5000 u 4 hrly and with continuous (C) I.V. heparin in comparable dosage with daily dosage adjustment designed to maintain activated partial thromboplastin times between 1½ and 2½ × control values. Patients with massive pulmonary embolism are excluded.Fifty-five patients have now received I heparin and 50 C heparin. Age and sex distributions, clinical diagnoses and factors potentially predisposing to hemorrhage were similar in the 2 groups. The clinical diagnosis was radiologically confirmed in 80 of the 105 patients. Although no patients have had a certain pulmonary embolus during treatment 5 in each group had suggestive clinical and/or radiologic findings. Pain or tenderness occurred at a new site or in an asymptomatic leg in 7 patients on I heparin and in 13 on C heparin. Hemorrhage occurred in 18 patients on I and 16 on C heparin. Major bleeding occurred in 7 patients and was spontaneous in 5 of these, in each group. Minor bleeding occurred in 15 patients on I heparin and 12 on C heparin. Some of these also had major bleeding. No patients have died of bleeding. One patient has died from progressive respiratory failure due to the original emboli. These preliminary results demonstrate that, although mortality is low, both hemorrhage and clinical features suggestive of recurrent thromboembolism are common during the first 10 days of heparin therapy. They also suggest’that continuous administration of heparin with laboratory control may have no significant advantages over the simpler intermittent constant dosage treatment regimen.
α2-Plasmin Inhibitor: A Novel Inhibitor of Blood Coagulation and Kinin Generation
Authors: Saito H. , Goldsmith G. , Moroi M. , Aoki N.
Published: May 2019

A novel α2-p1asmin inhibitor (α2Pl), chemically and immunologically distinct from any known inhibitors, has recently been isolated and characterized from human plasma (Moroi and Aoki, J. Biol. Chem. 251: 5956, 1976). We have studied the effect of purified α2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentrations, (50 μg/ml), α2PI inhibited the clot-promoting and prekal1 ikrein-activating activity of Hageman factor fragments (HFf, MW = 30,000), the amidolytic, kininogen-ase and clot-promoting activities of plasma kallikrein, and the clot-promoting activity of activated plasma thromboplastin antecedent (activated PTA, XIa). For example, activated PTA was inhibited to 50% and 12% of original activity after incubation with α2PI for 10 and 30 min at 37°c respectively. At higher concentrations (200 μg/ml), activatedStuart factor (Xa) was also inhibited. Heparin (1.5 units/ml) did not enhance the inhibitory function of α2PI against HFf, plasma kallikrein or activated PTA. These results suggest that α2PI is an inhibitor of broad specificity that may play an important role in regulation of blood coagulation, fibrinolysis and kinin generation.
Fibrinopeptide a Determination in Clinical Plasma Samples
Authors: Gerrits W.B.J. , Flier O.Th.N. , van der Meer J.
Published: May 2019

Since the development of radioimmunoassays for fibrinopeptide A (FPA), several studies have been reported on the levels of FPA in plasma from patients.Thus, elevated plasma levels of FPA have been described in disseminated intravascular coagulation, with or without consumption coagulopathy, venous thrombosis or pulmonary embolism. Increased levels of FPA have also been reported during pregnancy and in malignancies. In the majority of these patients, intravenous administration of heparin resulted in a normal FPA level, suggesting that the initially elevated FPA level was caused by the action of thrombin.However, in some patients heparin injection did not lead to normalization of FPA levels. The presence of thrombin in the blood circulation may lead to elevated FPA levels, but also to an accelerated in vitro generation of FPA, which occurs if an anticoagulant without heparin is used. Our studies demonstrate that an enhanced in vitro generation of FPA often occurs in blood samples from patients with elevated FPA levels, but also in samples with a normal FPA level. The mechanism responsible for the latter phenomenon is not clear.Possibly, proteases other than thrombin can lead to the generation of FPA.
Quantitative Estimation of Circulating Fibrinmonomer by Means of Affinity Chromatography on Fibrinogen-Agarose (FG-Ag)
Authors: Heene D.L. , Matthias F.R.
Published: May 2019

The presence of circulating fibrinmonomer and fibrinmonomer-complexes indicates indirectly the intravascular action of thrombin. The analytical detection of these fibrinogen derivatives is mainly achieved by means of three methods: the ethanol test, agarose-gel-chromatography and affi nity-chromatography on fibrinogen-agarose. The principle of the latter procedure is based on the property of fibrinmonomer to form complexes with fibrinogen according to the accessability of polymerization sites. This method (Heene, Matthias: Thrombos.Res. 2,137,1973) was investigated in detail with regard to its application on clinical cases with hypercoagulability and DIC. Studies on plasma samples containing 3H-labeled fibrinmonomer confirmed specific and selective adsorption on FG-ag and complete recovery following elution according to standard conditions of the chromatographic procedure. The absolute amount of fibrinmonomer in plasma of normal donors (n = 27) was found to be 0.7 mg/100 ml which is 0.25% of total plasma fibrinogen. In samples of patients with hypercoagulability (myocardial infarction, DIC) considerable increase of fibrinmonomer can be detected quantitatively even if other coagulation assays fail to reveal procoagulant activity. Comparative studies to the ethanol test indicate that a positive ethanol test can be expected if plasma fibrinmonomer concentration ranges from 7 to 11 mg/100 ml and above.
High Molecular Weight Fibrin(OGEN) Complexes
Authors: Alkjaersig N.
Published: May 2019

Plasma fibrinogen, because of catabolism of fibrinogen in vivo, exhibits biochemical heterogeneity, manifested by molecular weight and other differences between the various thrombin clottable moieties. These comprise high molecular weight fibrin(ogen) complexes (HMWFC) ranging in molecular weight from 400,000 to 1,000,000, native fibrinogen (m.w. 330,000) and derivatives of fibrinogen smaller than the native protein (m.s. 260,000 or less). These moieties may be assayed, in plasma as well as in the purified system, by gel exclusion chromatography with immunologic assay of chromatographic effluents by the Technicon Immunoprecipi-tator and mathematical analysis of elution profiles. The coefficient of assay variation for the individual moieties is 7%.

Normal control subjects show (mean ± 1 s.d.) 7.7 ± 6.2% (18.4 ± 21 mg%) of plasma fibrinogen in higher molecular weight complex form, 67 ± 10.3% monomeric fibrinogen and 25.8 ± 6.4% fibrinogen first derivative. Values for plasma HMWFC exceeding mean + 2 s.d. of normal (20% or 71 mg%) are classed as abnormal and regarded as demonstrative of enhanced fibrin formation. The hypothesis that enhanced fibrin deposition is associated with the presence of demonstrable pathology, extravascular or intravascular fibrin deposistion or thrombosis, has been validated in several human disease models.
Factors Affecting the Factor X Activator Activity of Human Platelets
Authors: Vermylen J. , Semeraro N.
Published: May 2019

Several recent studies have indicated that patients with a thrombotic tendency have enhanced platelet coagulant activity. It therefore is of importance to attempt to identify factors which modify platelet coagulant activities. Recent work in our laboratory has provided evidence that human platelets possess the capacity to directly activate factor X.Adenosine-5'-diphosphate, collagen, acetylsalicylic acid and prostaglandin E1 do not modify this activity. All endotoxins studied however clearly enhanced this activity.Furthermore, infusion of ‘activated’ prothrombin concentrates in haemophiliacs with or without factor VIII inhibitor enhanced the activity of this platelet activator of factor X. The hypothesis has been put forward that this may be the mechanism through which ‘activated’ prothrombin concentrates ‘bypass’ factor VIII of IX inhibitors. Platelet isolated from platelet-rich plasma to which the ‘activated’ prothrombin concentrate had been added at a concentration approaching the maximal concentration achieved following in vivo infusion, also showed an increase in platelet coagulant activity. Work is in progress to identify the component in ‘activated’ prothrombin concentrates which enhances platelet coagulant activity.
Stimulation of Cultured Endothelial Cell Proliferation and Metabolism by Platelets and Vasoactive Agonists
Authors: Shepro D. , D’Amore P.
Published: May 2019

Platelets via their secretions communicate and regulate endothelial cells (EC), which function as first level sensors in a biofeedback mgchanism to maintain blood and tissue fluid homeostasis. Platelets at a concentration of 105/mm3, serotonin (10-5M), thrombin (1 unit/ml), ADP (10-6M), catecholamines (10-6M) and histamine (10-6M) stimulate EC proliferation in culture from 150-1000 percent of controls. That agonists so diverse in form have a similar effect suggests the mobilization of a second messenger, such as calcium, as the common denominator. 45Calcium influx in cultured EC increased 5-24 times that of controls following incubation with platelets, platelet secretions, and substances known to circulate in blood following vascular injury. Increased adenine uptake was also observed in response to these agonists, which supports the premise of a regulating role for platelet secretions on EC.Lastly, the increased calcium flux was correlated with the induction of ornithine decarboxylase (ODC) activity. ODC is the rate limiting enzyme in the synthesis of polyamines that are theorized to regulate many cellular functions. ODC induction was 15 fold above baseline following stimulation with complete media, peaking at 3 hr. Preincubation with lanthanum chloride (LaCl3) inhibited this stimulation; the inhibition was reversed by the removal of the LaCl3. A similar response was obtained with 2 mM ethylene glycolbis. N,N'-tetraacetic acid in the media. These data support the validity of using EC in culture to simulate in vivo conditions, and the measurement of calcium flux, adenine uptake, and ODC activity, or combinations of the three, are significant parameters when cultured EC are used as an assay system.
Kinetics of Factor X Activation Catalyzed by Factor VII.
Authors: Nemerson Yale
Published: May 2019

Factor X activation is the point at which the intrinsic and extrinsic pathways of coagulation converge. Accordingly, events which control the rate of activation of Factor X might be central to the overall control of coagulation. We have therefore attempted to extract the kinetic parameters which govern this reaction. We have also devised and utilized a new assay for this reaction based on the release of radiolabeled activation peptide from Factor X. Utilizing Factor VIIa (2-chain VII) in the presence of saturating amounts of tissue factor, the KM was found to be 0.34 μ M and the Kcat 32 sec-1. The value for the KM is about twice the concentration of Tactor X in plasma. This means that rather small variations in Factor X concentration, such as those encountered in the normal population, would have significant effects on the rate at which Factor X is activated. We have also found that the effect of tissue factor on this reaction is mainly on the catalytic activity of Factor VII although in the absence of tissue factor there is also a 10-fold increase in the KM. Factor VII is predominantly present in plasma as a single polypeptide chain. Utilizing similar techniques we have also examined the reaction catalyzed by this form of Factor VII. All progress curves showed a prolonged lag followed by a sharp upward deflection. The mechanism underlying this phenomenon is now being investigated and will be discussed.
Structural Changes in Bovine Factor X Associated with Activation
Authors: Furie B. , Furie B.C.
Published: May 2019

Bovine Factor X is converted to Xa in the presence of Ca (II) by the coagulant protein of RVV. To monitor structural transitions in Factor X during conversion, the ultraviolet absorption, fluorescence emission, and circular dichroism spectra of Xa and Factor X were compared. The U.V. difference spectrum in the aromatic region comparing Xa and Factor X is characterized by differences due to tryptophan and tyrosine perturbations. The activation of Factor X yielded a time-dependent increase in this spectrum which was linear for about 60 min. and which paralleled the development of activated Factor X activity. The binding of Ca (II) to Factor X is associated with a red-shifted tryptophan difference spectrum; however, this perturbation makes only a small contribution to the total perturbation observed during Factor X activation. Solvent perturbation studies in 20% glycerol suggest that an average of 3.1 tryptophan residues and 9.0 tyrosine residues are exposed to solvent in Factor X; an additional 0.5 tryptophan residue and tyrosine residue become exposed to solvent during activation of Factor X. The activation of Factor X by the venom protein is associated with a small red shift in the intrinsic tryptophan fluorescence emission spectrum. Far- and near-U.V. circular dichroism spectroscopy detected no difference between Factor X and Xa. In summary, the activation of Factor X to Xa appears associated with exposure of tryptophan and tyrosine side chains previously buried within the protein and with minimal changes in the secondary structure. These results suggest that conversion of Factor X to activated Factor X involves functionally important, but structurally subtle, changes in the three-dimensional structure.
Blood Coagulation in the Horseshoe CRAB (Limulus polyphemus): A Model for Mammalian Coagulation and Hemostasis
Authors: Levin Jack
Published: May 2019

The amebocyte is the only type of circulating cellin the blood of Limulus. It is a nucleated cell, the cytoplasm of which is packed with granules. The coagulation system of Limulus is contained exclusively within the amebocytes.Furthermore, factors necessary for blood coagulation are localized within their cytoplasmic granules. Cell-free plasma does not clot and is not required for coagulation. Aggregation and disruption of cells follow exposure of amebocytes to foreign surfaces. Endotoxin produces similar changes and in addition, results in coagulation of the blood, following release from amebocytes of the components of the coagulation mechanism.Coagulation, produced by endotoxin, is the result of activation by endotoxin of an enzyme (or series of enzymes) that in turn reacts with the clottable protein. Endotoxin does not react directly with the clottable protein. The rate of the reaction depends upon the concentration of endotoxin.

In both Limuli and mammals, disruption of the integrity of the circulatory system or exposure of blood to foreign surfaces results in aggregation of amebocytes or piatelets, with subsequent changes in shape, disruption of granules, and release of cellular constituents into the surrounding environment. Both cells are necessary for hemostasis but in addition, amebocytes are necessary for the coagulation of blood. The presence of the entire coagulation system in amebocytes provides the basis for suggesting that coagulation in animals was initially a cellularly based function. The role of the amebocyte in controlling infection and its reaction to endotoxin suggest that the response of platelets and the blood coagulation system in various mammals to gram-negative infection or endotoxin is a remnant of this mechanism.
Hemostasis in Nonmammalian Vertebrates: An Overview
Authors: Dodds Jean W.
Published: May 2019

A common feature of comparative hemostasis throughout the animal kingdom is the participation of cell aggregation and plasma coagulation processes in the sealing of wounds. This applies to many invertebrates and all vertebrates. It is tempting therefore to assume a direct evolutionary progression from primitive invertebrates to advanced vertebrates. The major factors contributing to hemostatic plug formation and dissolution (vessel contraction, plasma coagulation, platelet or thrombocyte reactions and fibrinolysis) are present to greater or lesser extent in most nonmammalian vertebrates including Cyclostomes, Elasmobranchs, Bony Fishes, Amphibians, Reptiles and Birds. Of these functions, the thrombin-fibrinogen reaction has evolved as the final stage of the coagulation mechanism of all vertebrates. Prothrombin conversion to thrombin, accomplished by a tissue factor, is also common to all classes of vertebrates although the plasma factors involved in prothrombin activation have not been delineated for many lower forms of vertebrates. Similarly the exact relationship between the various types of cells involved in hemostasis has yet to be established. Although mammalian platelets probably were derived from invertebrate and lower vertebrate thrombocytes, this is yet to be proven. The challenge remains to develop a more thorough understanding of the basic mechanisms of hemostasis and thereby provide additional biochemical evidence for evolutionary progression.
Modification of Thrombin-Induced Platelet Aggregation by Estrogen
Authors: Nagasawa H. , Steiner M. , Baldini M.
Published: May 2019

The effect of estrogens on platelet function has remained a subject of considerable controversy. Neither in vivo nor in vitro studies have yet established a basis for a possible mode of action of this hormone on platelets. Our studies were predicated on previous results obtained by one of us suggesting a direct interaction of estrogens with antithrombin III(AT III), Platelets were isolated by conventional method from freshly drawn blood of volunteers, washed twice with Ca2+-free Tyrode buffer and finally suspended in this medium at a concentration of 4.5 × 105 platelets/mm3. Aggregation was induced by addition of 0.01 units of purified bovine thrombin (390 NIH units/mg protein). Aggregation was immediate reaching a maximum within 1.5-2 min.AT III purified from human plasma (2 U/mg protein) inhibited thrombin-induced aggregation in a predictable, concentration-dependent manner. Addition of 0.06 U AT III produced almost complete inhibition. The inhibiting effect of AT III was found to be related to the platelet concentration. Increasing the latter diminished progressively the effect of AT III on thrombin-induced aggregation.Beta-estradiol also inhibited the AT III effect on thrombin-induced aggregation abolishing it at a concentration of 5 × 10-6M. The minimal concentration of β-estradiol which produced a recognizable effect in this system was 5 × 10-9M. These results indicate a direct effect of estrogen on AT III, modifying the protein in such a way that subsequent interaction with thrombin either becomes impossible or does not lead to the inactivation of the enzyme. In addition a possible neutralization of AT III by intact platelets is suggested from our data.

These studies were supported by a contract from the AEC.
Platelet-Mediated Vasoconstriction: Evidence in Vivo
Authors: Baumgartner H.R. , Tschopp Th.B.
Published: May 2019

Serotonin, PG-endoperoxides and thromboxane A2 - released from platelets by collagen for example - contract vascular smooth muscle in vitro. Platelets adhering to subendothelium in vivo undergo the release reaction. Platelet adhesion to subendothelium and concomitant vasoconstriction were therefore measured morphometrically in cross sections of rabbit iliac arteries which had been fixed by perfusion of glutaraldehyde at 110 mm Hg: (l) At 1.25, 2.5, 5, 10 and 20 min after complete denudation of endothelium by balloon catheter injury; (2)l0 min after partial denudation; (3)l0 min after complete denudation in rabbits made thrombocytopenic by injection of a heterologous antibody against platelets. The non-ballooned iliac artery served as control. In sham operated rabbits the vessel diameter (D) as derived from the length of the internal elastic lamina in cross sections was similar for both iliac arteries. (l)l0 min after denudation - when surface coverage with platelets approached 100 % and aggregation was maximal - D was reduced by 28+2% (mean ± SE, η = 20, 2p < 0.001). At 1.25 or 20 min - when few platelets or only degranulated platelets, respectively, adhered - D of ballooned and control arteries were again similar. (2) Localized platelet adhesion caused localized vasoconstriction. The extent of platelet adhesion and vasoconstriction correlated (r = 0.64, n = 44, 2p < 0.001). (5) In thrombocytopenic rabbits surface coverage with 22±7% platelets was associated with reduction of D by only 10+3%.

Thus platelets freshly adhering to subendothelium in vivo appear to induce a transient contraction of medial smooth muscle cells during a few minutes.
Inhibition of Factor XI by Myeloma Protein M.
Authors: Rimon A. , Raz I. , Lahav M.
Published: May 2019

Waldenstrom’s macroglobulinemia sometimes involves hemostatic disorders. It has been assumed that this may be due to interaction of the myeloma protein with soom blood coagulation factor(s). In the present investigation the sera of seven myeloma patients were studied for a possible interaction between the myeloma protein and clotting factors II, VIM, IX and XI.

Normal human plasma was incubated for 30 mi η at 37°C with different dilutions of patients’ serum or of purified myeloma proteins. The ability of this mixture to restore the clotting times of various deficient plasmas was tested by the partial thromboplastin time test.

Factor XI activity of normal plasma was substantially inhibited upon incubation with patient’s serum or with purified myeloma protein M. This effect seems to be specific for myeloma protein M since it was neither observed with myeloma proteins G or A, nor with normal IgM. Other coagulation factors investigated so far were not inhibited by myeloma protein M. It is concluded that this protein is specifically affine to factor XI resulting in hindering the latter’s activity.
Evaluation of the Blood Compatibility of Biomaterials by Plasma Kallikrein Derived from Contact Activation
Authors: Becker U. , Fuhge P. , Heimburger N. , Fischer P.
Published: May 2019

Contact activation is considered as the crucial step to trigger blood coagulation on artificial surfaces. It is characterized by the conversion of proenzymes in plasma to active enzymes. Kallikrein is one of the proteinases which are generated in the course of contact activation. Consequently, its proteolytic activity can be used for monitoring the velocity of contact activation. This is demonstrated by recording the cleavage rate of the synthetic chromogenic substrate Bz-Pro-Phe-Arg-pNA. Polymer surfaces are known to be only weak activators of the contact phase. Therefore, the aim of the study was to elaborate a system sensitive enough to differentiate between various polymer surfaces. Two different approaches are shown to be effective:

1, In a one-step assay diluted plasma is incubated with the materials to be tested in the presence of the chromogenic substrate.

2. In a two-step assay whole blood or undiluted plasma is extensively incubated with materials before the residual contact activation capacity is determined by addition of ellagic acid.

The release of kallikrein is shown tobe specifically surface mediated by using plasma deficient in single clotting factors and by its sensitivity against various inhibitors. The methods were applied to the evaluation of biomaterials of different shape like beads and tubings. The results correspond to those obtained with coagulation tests. Thus the method may provide a useful tool for the development of blood compatible biomaterials.
Studies on Components of the plasma kallikrein-Kinin System in Normal Subjects and Patients with Septicemia
Authors: Aasen A.O. , Gallimore M.J. , Lyngaas K. , Larsbraaten M. , Amundsen E. , Eriksen N. Smith
Published: May 2019

Over the past decade evidence that the plasma kallikrein-kinin system is activated during septicemia has accumulated. We have used several tests, including newly developed chromogenic peptide substrate assays, to study components of this proteolytic enzyme system in plasma samples from normal subjects and patients with septicemia. The parameters studied were Hageman factor(HF), plasma kallikrein (KK), plasma prekallikrein (PKK), high molecular weight kininogen(HMwK), functional kallikrein inhibition (KKI), c1-esterase inhibitor (CIINH) , α2-macroglobulin (α2 M)and α1-antitrypsin (α1AT ). In samples from patients with fatal sepsis, levels of HF, PKK, HMwK, α2M and KKI were all markedly reduced and spontaneous KK activity was detected. CIINH and α1AT levels were much higher than normal. In plasma samples from three patients with septicemia who subsequently recovered mean plasma levels of PKK, KKI, HMwK and CIINH were higher than in the samples from the patients who died. Our results emphasize that the plasma kallikrein-kinin system becomes activated during septicemia and suggest that functional kallikrein inhibition contributes to survival.
Factor VIII Related Antigen-Activity Differences in Intrauterine Growth Retardation
Authors: Whigham K.A.E. , Howie P. W. , Shah M. , Prentice C.R.M.
Published: May 2019

Haemostatic values, hormone and uric acid levels were determined in women during pregnancies suspected of growth retardation, and compared with normal pregnancies. Nine of the 21 suspect pregnancies resulted in a small baby, below the 10th weight percentile. Platelet count, fibrinogen, soluble fibrin complexes and fibrinogen degradation product values were not significantly different between the normal and growth retarded group. In normal pregnancies. F VIII related antigen (VIII RAg) and clotting activity (VIII C) increased with gestation up to 36 weeks, with antigen about 20% higher than activity. In mothers who delivered growth retarded babies the VIII RAg minus VIII C difference (or VIII RAg/VIII C ratio) was significantly greater. Seven of the nine mothers who delivered small babies had a VIII RAg-VIII C different greater than 125% (VIII RAg/VIIIC ratio greater than 1. 75), while only one of the 12 mothers who delivered normal weight babies had this difference. Serum prolactin, oestriol or uric acid levels provided no further means of distinguishing between the two groups. The VIII RAg/VHI C ratio appears to be the single best test for distinguishing growth retarded babies from normals in a clinically suspect group.
The Role of Prostaglandins in Platelet-Vessel wall Interactions
Authors: Boutgain R.H. , Six F. , Potvliege P.
Published: May 2019

In an in vivo model developed for the study of arterial thrombosis in the white Wistarrat, it was demonstrated that superfusion of the investigated arterial segment with cyclo oxygenase inhibitors (ASA, indomethacin, flurbiprofen) resulted in a marked decrease of ADP-induced thrombosis while tranylcypromine enhanced the thrombotic phenomenon. As other MAO inhibitors were without effect, it was concluded that tranylcypromine possibly decrsibsed the PGE2-synthetase activity of the vessel wall. In the light of these findings we then suggested that the platel et-vessel wall interaction was dependent at least to some extent on the cyclic endoperoxides-PGI2 ratio of the endothelium and eventually of the deeper vascular structures. Electron microscopic studies on arterial segments used in our model revealed that suloctidil markedly decreases smooth muscle cell hypertrophy following local deendothelialization. Furthermore reconstitution of the endothelial lining consisting in endothelial cell hypertrophy and sliding over the dcendothelialized are until cell contact is made was also delayed. Following the intravenous administration of suloctidil (0.5 mg/kg) in our model the thrombus enhancing effect of locally superfused tranylcypromine was Offset. These experiments suggest that a decreased PGI2-synthetase activity is inhibited by a platelet function active drug such as suloctidil.
Demonstration of Antithrombin III in Fresh and Cultured Endothelial Cells from Human Umbilical Cord
Authors: Chan Vivian , Chan T.K.
Published: May 2019

We have shown by immunofluorescent technique that the distribution of antithrombin III (ATIII) in human tissues was concentrated around the microvasculature of the lungs and kidneys, as well as veins and small arteries of other organs (liver and spleen). It would seem that ATIII is stored and/or synthesized in the endothelial cells similar to Factor VIII-RAG and Plasminogen Activator. Endothelial cells were isolated from human umbilical cord by collagenase and cultured according to Chemethod described by Shearn etal (1977). In freshly isolated endothelial cells, ATIII could be demonstrated by indirect immunof1uorescent technique and radio immunoassay confirmed the presence of 14.8 ng per 106 cells. After 7 days’ culture, the supernatant from 106 cells contained about 15 ng and the cultured cells (106) contained 16.9 ng ATIII. The presence of ATIII in cultured cells was also confirmed by the positive immunofluorescence. Hence the endothelial cells play an active role in preventing thrombosis by the synthesis and liberation of ATIII, the major natural inhibitor of the intrinsic pathway of Coagulation.

Reference: Shearn S.A., Peake I.R., Ciddings J.C., Humphrys J. and Bloom A.L. Thrombosis Research, 11, 43, 1977.
Metabolism of Adenosine Diphosphate by Cultured Umbilical Endothelial Cells
Authors: Dosne A.M. , Escoubet B. , Legrand C. , Bodevin E.
Published: May 2019

Vascular endothelium has been considered to be responsible for the clearance of circulating adenylnucleotides. Endothelial cells were collected from human umbilical veins and as Pnmary cultures. The cells were incubated in Medium 199 pH 7.4, with 8[14C]-ADP 10-5M and the metabolites were separated by thin-layer chromatography from the supernatant and from the cellular perchloric extract.

ADP was degraded as an exponential function of time into AMP and then into adenosine, which was the main catabolite found in the medium at 30 min. Adenosine was taken up by the cells at a constant low rate and then transformed into ATP. The rate of ADP degradation depended upon the physical state of the cells and was increased by cell dissociation The half life of ADP was S min in presence of adherent cells and 3 min in presence of col 1agenase-dissociated cells. The ADP clearance increased as an exponential function of cell density but decreased with the culture age (5 up to 13 days).

These results suggest that endothelium may control the formation of circulating adenosine, which contributes to local blood flow regulation and to the non-thromboqenic properties of endothelium.
Detective Platelet Surface Markers in Chronic Myeloid Leukemia (CML)
Authors: Vainer H. , Bussel A.
Published: May 2019

Leukemic platelets (LP) from patients suffering from CML were shown to express altered surface membrane glycopoteins (Vainer s Bussel, 1977, Intern. J. Cancer, 19, 143). The present study wax devoted to the examination of other platelet surface components in 8 patients with CML, before any treatment : i) UDP-Galactose: glycoprotein galactosyl-transferase (GTase) activity, evaluated from the amount of radioactivity incorporated a) into the acid-precipitable material and b) In the labeled soluble reaction products, eluted following autoraduichromatography - and ii) 14C epinephrine (E) binding sites on isolated LP membranes, determined by a filtration procedure.

It was found that i) LP can catalyze the transfer of 14C-Galactose from its activated form (UDP-Gal) onto endogenous and exogenous (ovomucoid) acceptors. The transfer reaction in LP was found at a rate 2 to 4 fold greater than in normal platelets (NP), without affecting the values of the apparent Km for UDP-Gal or other kinetic parameters, ii) Isolated membranes from LP specifically bind 14C-E to alpha adrenergic type sites, but the number of receptors for E, available on LP membrane was lower (or present in a cryptic form) than the level reported in NP (Vainer H., 1977, Cell Str. & Function 2, 267).

The modulation of both GTase activity level and E receptor number in LP from CML patients could be involved in or correlated with their abnormal function and morphology.
Hemostatic Disorders and Platelet Nucleotide Release in Myeloproliferative Disease
Authors: Hagedorn A.B. , Bowie E.J.W. , Owen C.A.
Published: May 2019

Since patients with myeloproliferative disorders may have bleeding tendencies, the surgeon, in particular, is anxious for an hemostatic evaluation if splenectomy is contemplated. It is known that platelet aggregation, particularly with epinephrine, tends to be reduced in these patients. The nucleotide content of their platelets may be deficient. Furthermore, megakaryocytic fine structure is often abnormal. We have studied, In detail, 9 patients with hemostatic disorders. Diagnoses included polycythemia vera, agnogenic myeloid metaplasia, evolving myeloproliferative disease and erythroleukemia. Ages ranged from 36 to 75 years. Bleeding tendencies, including bruising, operative or postoperative bleeding, melena, hematuria, and hemarthrosis, characterized 8 of the 9 patients; the one exception had normal platelet ADP and elevated ATP. All had abnormal platelet aggregation, but the extent of the abnormality could not be related to the ADP and ATP contents of the platelet.ADP (normal 26.7 ± 6.5 nmol/109 platelets) was reduced in 7. ATP (normal 38.6 ± 7.6 nmol/109 platelets) was reduced in 1, elevated in 2 and normal in the other 6. In no patient were both values normal. Nucleotide release induced by collagen activation was measured in 6 of the patients. In all 6 it was deficient whether platelet ADP were normal (1 case) or depressed (5) and whether platelet ADP were elevated (1) or decreased (3).
Platelet Associated IgG in Chronic Hepatitis
Authors: Landolfi R. , Fedeli G. , Storti S. , Laghi F. , Leone G.
Published: May 2019

Raised levels of platelet-associated IgG have been described in many pathological conditions such as ITP, LES, rheumatoid arthritis, lymphoma, drug thrombocytopenia. We measured surface-bound platelet IgG in 60 patients suffering from chronic hepatitis. In all cases diagnosis was confirmed by needle biopsy of the liver and by pertinent laboratory data; the technique used was that described by Dixon et al.(1975). In chronic active hepatitis (40 cases) we found increased amounts of platelet-associated IgG with values ranging from 110 to 480 ng/107 platelets (mean 195 ng; < 0.O01). In persistent hepatitis (40 cases) surface-bound platelet IgG were always less than 70 ng/107 platelets. A significant inverse correlation between increased amount of platelet IgG and the platelet count was not found. This test can be of considerable help in the differential diagnosis of chronic hepatitis as well in the follow up of these patients for prognostic and therapeutic decisions.