bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibrinogen, Fibrinogen Heterogeneity and Fibrinolytic Activity in Diabetes Mellitus
Authors: Gurewich Victor , Lipinska Izabella , Pulini Maria , Gordon Edwin , Lipinski Boguslaw
Published: May 2019

Fibrinogen (F) concentration, fibrinogen heterogeneity on 3.5% Polyacrylamide gels, fibrinolytic activity (FA) measured by euglobulin fraction on fibrin plates and fibrin degradation products (FDP) were measured in 66 patients with well documented diabetes mellitus (DM) and in 50 healthy subjects of comparable age. A high molecular weight and 2 lower molecular weight (LMW and LMW1) fibrinogen fractions were identified. The mean values and statistical evaluation of their differences were as follows:

The clinical duration of DM, degree of control or type of medication did not appear to influence these findings. However, within the patient group, those with clinical evidence of microvascular disease had significantly (p<0.02) higher LMW1 fibrinogen and lower FDP (p<0.01) than the remainder. These findings suggest that DM is associated with fibrin deposition, and accelerated F degradation to LMW and LMW1 fractions and that these processes may be associated with the development of vascular lesions.
Fibrinolytic System-Heparin Interaction
Authors: Villaverde C.A. , Badimón L. , Bastida E. , Valdecasas F.G.
Published: May 2019

It was studied in vitro the effect of Heparin(Hp) directly on Urokinase(UK) induced fibrinolysis, Hp on rat euglobulins(Eu)-UK activated, and Hp on whole rat blood UK-activated (through the fractions: whole and diluted blood, whole and diluted plasma, Eu and inhibitors). For testing the influence of Hp with UK we used the polyvalent method on fibrin plate that we presented in the X International Congress of Angiology, Tokyo, Sept. 1976. We have assayed UK concentrations from 0.25 to 100 Pl.U./ml. and Hp concentrations from 0.001 to 1000 I.U./ml. It was not found significantly modifications in the Hp effects on UK. In the system Eu-UK-Hp we have obtained decrease in fibrinolytic response of Eu-UK activated with every Hp concentration. Studying the above mentioned hematic fractions, the results were different related to the Hp concentration, thus the low dose produced increase of fibrinolytic activity, while higher dose produced clear inhibition in each fraction. Assaying isolated inhibitors UK-mixed, in order to quantify the fibrinolytic response in Hp presence, we found a progressive increase of the inhibitory potential accordingly with the increase of Hp concentration. In rat, in vivo, we obtained significantly inhibition in every tested blood fraction with Hp higher dose.
Biological Properties of an Heparin-Poly (Methyl Methacrylate) Copolymer
Authors: Labarre D. , Boffa G.A. , Jozefowicz M. , Boffa M.C.
Published: May 2019

The heparin-poly(methyl methacrylate) copolymer was prepared by polymerization of methyl methacrylate on an heparin radical. It was initiated by cerium IV ions and heparin in nitric aqueous solution.

An heparin effect was present in the plasma in contact with the copolymer. It was not due to a release of heparin in the plasma, but to a contact effect. The anticoagulant effect was depending upon the concentration of the copolymer. The activity of clotting factors in plasma, after a contact with the copolymer was not decreased except for factor V. Antithrombin III was selectively adsorbed on the surface of the copolymer.

The copolymer lost progressively its anticoagulant properties after each contact with the plasma. This might be due to the adsorption of fibrinogen on the copolymer. Labelled 125I albumin, transferrin, Ig G and fibrinogen were not similarly desorbed in the presence of serum and of plasma.

This copolymer seems to offer interesting properties for blood devices.
Analysis of Anticoagulation Activities of Heparins - Is usp Heparin Assay Reliable?
Authors: Shen Linus L. , Barlow Grant H.
Published: May 2019

The in vitro “over-all” activity of heparins was analyzed by the technique of rigidity measurements with plasmas upon recalcification. This technique allows the measurement of the entire clotting process of a heparinized plasma, and is thus more sensitive and reliable than clotting time determination. Contrary to its action to the clotting of whole blood, heparin reduces the final rigidity of a plasma clot only when the degree of heparinization exceeds certain limit. A log-log plot of clotting half-time vs. amount of heparin may be used to compare the anticoagulation activity of various heparins in human plasma and in sheep plasma. Results show that heparin with molecular weight around 20,000 possesses highest specific activity, the activity drops sharply when molecular weight increases or decreases. The anticoagulation activity of heparin in human plasma, expressed by the increase in clotting half-time is up to 100 times more effective than that in sheep plasma but responds less sensitively to the change in heparin concentration. Using the same heparin standard, the specific activity of certain heparin fractions assayed in human plasma differs from that assayed in sheep plasma. The discrepancy increases with the decrease in heparin molecular weight. The discrepancy was also observed with some heparins of different tissues and sources. The USP heparin assay, which uses sheep plasma as the assay medium, therefore does not necessarily reflect the true activity in human blood clotting system.
The Heparin Inhibiting Property of Brain Thromboplastin
Authors: Gomperts E.D. , Zucker M.
Published: May 2019

Antithrombin III is one of the serine proteinase inhibitors of the plasma which has been shown to specifically inhibit thrombin as well as Factor X. Heparin acts via antithrombin III, the heparin cofactor, hence it is difficult to explain the relative insensitivity of the prothrombin time to the presence of heparin in plasma as both thrombin, ana Factox Xa are associated functionally with the prothrombin time. This insensitivity becomes more obvious on appreciating the extreme sensitivity to heparin of the activated partial thromboplastin time as well as the thrombin time. This communication reports the demonstration of heparin inhibiting action of brain thromboplastin. The response of the prothrombin time to heparin under various conditions, and the effect of brain thromboplastin obtained from various sources and by different preparative techniques on the action of heparin in vitvo have been studied. The heparin inhibiting activity was shown to parallel the tissue factor activity. It is heat labile, non-dialysable, destroyed by detergent activity and lies in a high molecular weight fraction of the brain thromboplastin preparation (>300,000). In addition to explaining certain in vitro phenomena, these observations may explain the previously observed heparin resistance in the generalised Schwartzman phenomenon.
Interlaboratory Oral Anticoagulant Quality Assessment by the Netherlands Federation of Thrombosis Services
Authors: van Dijk-Wierda C.A. , Hermans J. , Loeliger E.A. , Roos J.
Published: May 2019

The 50 laboratories of the Netherlands Federation of Thrombosis Services have participated since 1974 in a voluntary external and internal quality control program. The external program comprises a monthly distribution to the member laboratories of a series of artificially prepared control blood samples, two of which are identical. The overall variation of the coagulation times found were 10% (CV) in 1974 and 8% (CV) in 1975 and 1976. Performance improved rather abruptly at the beginning of 1975, after the application of a tight methodological standardization and improvement by the manufacturer of the thromboplastin preparation (Thrombotest) used by the great majority of the laboratories involved. The main source of variation wasfound to be random error in the Thrombotest determination, approximating 6%. interbatch variation of Thrombotest and inter-aliquot variation of control blood samples both do amount to approximately 3%.
The Platelet Dense Tubular System: Its Relationship of Prostaglandin Synthesis and Calcium Flux
Authors: Gerrard Jonathan M. , White James G.
Published: May 2019

Evidence that the dense tubular system (DTS) is the site of platelet prostaglandin synthesis derives from several observations. First platelet peroxidase is localized in the DTS. Aminotriazole which inhibits the platelet peroxidase, inhibits prostaglandin biosynthesis at the same concentration. Secondly, a similar peroxidase occurs in the secretory cells of the sheep vesicular gland (SVG) and other cells known to be involved in prostaglandin synthesis. Third, the DTS is smooth endoplasmic reticulum (SER) and SER, which is abundant in the prostaglandin synthesizing columnar cells of the SVG secretory units, is known to be involved in lipid synthesis and metabolism in other tissues. Evidence that calcium is stored in the DTS derives from ultrastructural studies showing that the DTS is analagous to the sarcotubules of skeletal muscle, and that the DTS has the capacity to bind divalent cations. Evidence that calcium flux and prostaglandin synthesis are closely linked comes from several observations which suggest 1) that movement of calcium ions to the site where arachidonic acid (AA) is released from the precursor phospholipids to be used for synthesis of prostaglandins, stimulates the AA release and 2) that synthesized prostaglandin G2, prostaglandin H2, and/or thromboxane A2 initiate platelet contraction by moving calcium from the site of synthesis to the vicinity of the contractile actin and myosin. The results suggest that the DTS, prostaglandin and thromboxane synthesis, and calcium flux are integral parts of the system modulating platelet activation.
Plasma High-Molecular-Weight and Low-Molecular-Weight Kininogens: Their Structures and Physiological Functions
Authors: Iwanaga S. , Kato H. , Han Y.-N. , Hashimoto N. , Sugo T. , Fujii S.
Published: May 2019

Bovine plasma contains two kininogens. One of them, HMW kininogen, has recently been characterized as a new coagulation factor, which is required for the activation of Hageman factor-mediated pathway. To elucidate the structural differences between HMW and LMW kininogens, these were fragmented with plasma kallikrein and snake venom kininogenase, and the resulting products were chemically identified. The kallikrein simultaneously released bradykinin and fragment 1·2 from HMW kininogen. Fragment 1·2 was a histidine-rich glycopeptide consisting of 110 amino acid residues, and its whole sequence and the location of the sugar moieties were established. The venom kininogenase liberated only bradykinin from LMW kininogen. Kinin (and fragment 1·2)-free proteins consisted of heavy and light chains. These chains constituted, respectively, the NH2-and COOH-terminal portions of both parent molecules and were held by a single disulfide bridge. The detailed studies on these chains indicated that the NH2-terminal portions of HMW and LMW kininogens are very similar for each other, whereas their COOH-terminal portions differ significantly. Concerning the results that HMW kininogen overcomes impaired coagulation and fibrinolysis of the kininogen deficient plasmas, and the kinin-free HMW protein has a weak corrective activity, it seems that the histidine-rich region found only in HMW kininogen may be closely related to the functions of the kininogen.
Antithrombin III and its Interactions with Heparin
Authors: Rosenberg R.D. , Jordan I.R. , Armand G. , Lam L.H. , Beeler D.L.
Published: May 2019

Heparin functions as an anticoagulant by binding to antithrombin and accelerating the rate at which this protein inhibitor inactivates the serine proteases of the hemostatic system. Despite considerable effort, the precise relationship between the structure of this mucopolysaccharide and its biologic properties has remained elusive.

We have provided the first evidence that only a small portion of a given heparin preparation binds tightly to antithrombin and is responsible for the bulk of anticoagulant activity. The major fraction of these products has very little anticoagulant effect.

In our presentation, we shall discuss optimal techniques for fractionating heparin into “highly active” and “relatively inactive” molecular forms.Furthermore, we shall consider the distinctive interactions of these two types of mucopolysaccharides with antithrombin, thrombin and other hemostatis system components.Lastly, we shall provide evidence that several structural differences exist between these two forms of heparin.
In Vitro Characterization of Rabbit Thrombin, Antithrombin III, and Thrombin-Antithrombin III Complex, and Determination of Their Survival Times in Vivo
Authors: Vogel Christine N. , Henry Kingdon S. , Lundblad Roger L.
Published: May 2019

Our intention is to study the interaction of rabbit thrombin with antithrombin III (AT-III) in vitro and in vivo. After activation of crude prothrombin with tissue thromboplastin and CaCl2, thrombin was purified and showed two species of thrombin with molecular weights of 36,000 and 39,000 daltons as determined by sodium dodecyl sulfate discontinuous gel electrophoresis. Rabbit AT-III was purified using a heparin agarose column and had a molecular weight of 55,000 daltons. The inhibition of thrombin by AT-III was followed by fibrinogen clotting assays and an AT-III-thrombin complex was observed on gel electrophoresis. For the in vivo studies both thrombin and AT-III were radiolabelled with Na125i using the solid state lactoperoxidase method and retained 99% of the pre-iodinated specific activity. Radiolabelled thrombin and a radiolabelled AT-III-thrombin complex were injected into different rabbits. The rate of removal of both was very similar with a half-life of approximately 9 hours. When radiolabelled AT-III was injected, the half-life was approximately 60 hours. Since the disappearance rate of thrombin more closely approximates that of the preformed AT-III-thrombin complex and is clearly shorter than the turnover rate of AT-III, the possibility is raised that thrombin combines in vivo with a native inhibitor such as AT-III and may in fact be removed from the circulation as a complex rather than as a native molecule.
Relationship Between Platelet Density and Number of Dense Bodies
Authors: Rendu F. , Lebret M. , Caen J.P.
Published: May 2019

Dense bodies are the storage site of 5-HT and their number might correlate with the different platelet populations. Mepacrine, a basic fluorescent dye, is a suitable tool for studying abnormalities in platelet 5-HT storage.

The content of platelet DB can be readily estimated by examination under fluorescence microscope of platelets charged with mepacrine. In human platelets, a mean number of 6.3 DB/ platelet was found. This estimation has been performed in platelets of different densities separated on sucrose gradient : heavy platelets, in which 5-HT uptake is strongly inhibited by reserpine, had a mean number of 7 DB/platelet and none was empty of DB ; light platelets which are less active in regard to 5-HT and insensitive to reserpine, had less than 3 DB/platelet and most of them had a cytoplasmatic fluorescence. Thus the proportion of light and heavy platelets can be directly established by staining platelets with mepacrine.

In view of this, platelets of two patients with an abnormal density distribution were studied for 5-HT and charged with mepacrine. The first presented an Hermansky-Pudlak syndrome (HPS), an increased proportion, of light platelets, a low 5-HT uptake insensitive to reserpine and a great reduction of mepacrine-stained DB. The other patient with a Bernard-Soulier syndrome had only heavy platelets ; 5-HT uptake in these large platelets was markedly increased and all platelets had more than 20 DB stained with mepacrine.

These results confirm that there is a good relationship between platelet density and number of dense bodies.
Normalization of Platelet Survival in an Experimental Model after Platelet Exposure of Thrombin in Vitro or adp in Vivo
Authors: Meuleman D.G. , Buchanan M.R. , Giles A.R. , Hirsh J.
Published: May 2019

It is generally assumed that when platelets are damaged during preparation, their subsequent survival in the circulation is decreased. We have examined the effect of exposure of platelets to stimuli such as thrombin and ADP prior to studying their interaction with either damaged vessel wall or in vivo aggregation by infused collagen. Catheters inserted in the abdominal aorta of rabbits produced marked vascular damage. This was associated with a significant shortening of platelet half life (T½) of homologous 51Cr-labelled platelets (T½-18hrs: Control animals T½-34hrs). In contrast, the platelet half lives of platelets degranulated by thrombin in vitro and those harvested from donors treated with ADP in vivo were 23 and 30 hrs respectively. In vivo collagen-induced platelet aggregation of the thrombin degranulated and the ADP exposed platelets was also found to be significantly diminished when compared with those obtained from untreated control animals.

These results suggest that exposure of platelets to either exogenous or endogenous stimuli could possibly mask a reduction in platelet survival in thrombovascular disorders and its modification by drugs.
Subcellular Localization and Secretion of Factor V by Human Platelets
Authors: Pifer D.D. , Colman R.W. , Chesney C.Mcl.
Published: May 2019

Previous studies have suggested that factor V in platelets is derived from plasma. In order to test this hypothesis, we studied the subcellular localization, release by aggregating agents, molecular size, and stability of factor V in human platelets. When platelet homoge-nates were fractionated on sucrose density gradient the factor V activity distributed primarily into the granules (0.076 units factor V activity/mg protein) with less amounts in the membrane fraction (0.014 units/mg protein) and virtually none in the dense granules or cytosol. Collagen released 79-3% of total platelet factor V clotting activity while ADP and epinephrine failed to liberate factor V activity, further supporting significant localization in the lysozomal granules rather than the dense granules. Platelet factor V can be solubilized by 0.1% Triton and has an apparent molecular weight of 470,000 as determined by 4% agarose gel filtration. In contrast, plasma factor V in the presence or absence of Triton has a molecular weight of 300,000. Factor V activity from the platelet homogenate exhibited biphasic decay at 37°C, suggesting more than one form, whereas plasma factor V shows a first order decay curve. These data suggest that platelet factor V is predominantly localized in the granules and is an intrinsic constituent of the platelet with different properties from plasma V.
Different Requirements for Intrinsic Factor-Xa Forming Activity and Platelet Factor 3 Activity and Their Relationship of Platelet Aggregation and Release
Authors: Walsh P.N. , Lipscomb M.S.
Published: May 2019

Platelets provide coagulant activity in part by making available membrane phospholipids which promote the reactions of: (1) factors XIa, VIII, IX and calcium to activate factor X (intrinsic factor-Xa forming activity or XaFA); and, (2) factors Xa, V and calcium to activate prothrombin (platelet factor 3 activity or PF3A). We have studied the availability of XaFA and PF3A, platelet aggregation and [14C]-5HT release in washed platelet suspensions, incubated with collagen.XaFA developed rapidly (100% activity at 15-30 sec) well before maximum [14C]-5HT release (2-4 min) and decayed to <10% at 5-10 min.PF3A developed slowly (100% activity after 20 min stirring with collagen) well after maximal aggregation and release. Maximal XaFA availability occurred in unstirred platelet suspensions incubated with collagen and not undergoing aggregation or release, and was not enhanced by stirring, whereas PF3A did not become available unless platelet suspensions were stirred with resultant aggregation and release.PF3A, XaFA, aggregation and release were inhibited by pretreating platelet suspensions with p-chloro-mercuribenzene sulfonate, concanavalin A or a combination of 2-deoxyglucose and antimycin A, whereas pretreatment with indomethacin inhibited aggregation, release and PF3A availability but not XaFA availability. These results indicate that the biochemical determinants of XaFA and PF3A are different.PF3A developed only when aggregation and release occurred whereas XaFA was independent of aggregation and release.
Studies Involved in the Development of a Specific Radioimmunoassay for Plasma Prothrombin.
Authors: Hassouna H. , Seegers W.H.
Published: May 2019

Antibodies directed against purified prothrombin, its activation products profragment 1 and profragment 2, its intermediate derivative prethrombin 1, and its enzyme portion thrombin were raised in rabbits. Antigenically reactive regions of each purified product were screened for crossreactivity and each antiserum tested for enzyme inhibition activity. Monovalent antisera to the native molecule have populations of antibodies that react with the purified and the iodinated I125 profragment 1, profragment 2, prethrombin 1, but not with thrombin. Profragment 1, profragment 2, and prethrombin 1 have one similar antigenic determinant site in common.Anti-sera to prethrombin 1 also crossreact with thrombin, implying the unmasking of a determinant site. Fresh oxalate and citrate plasma was tested on immunodiffusion against all specific antisera. The intact prothrombin molecule and each one of its reaction products, including thrombin, was found in plasma; they can be identified separately by their differing mobilities on Immunoelectrophoresis. Activation of prothrombin in plasma is inhibited by incubation of plasma with antibodies to the native molecule or to profragment 1, but not to prethrombin 1 or profragment 2. Purified thrombin added to its antiserum loses its activity on fibrinogen. Effect of addition of crossreacting fragments on the binding of I125 prothrombin to its antiserum was studied. The response curve of profragment 1 is identical to that of prothrombin; profragment 2 and prethrombin 1 inhibit only 10% of binding, thrombin has no effect. To avoid nonspecific effects, standard curves and incubates are run in 1/10 dilutions of prothrombin-free plasma. Prethrombin 1 and thrombin are assayed in a carefully dialyzed barium carbonate adsorbed plasma.
The Conversion of Bovine Abnormal Prothrombin into Prothrombin
Authors: Vermeer C. , Soute B.A. M. , Hemker H.C.
Published: May 2019

In order to characterize the enzyme system which converts abnormal prothrombin (PIVKA-II) in prothrombin we prepared liver cell-free extracts from normal healthy cows. The enzyme system devoid of either precursor or prothrombin was isolated by differential centrifugation and partly purified with the aid of column chromatography.

The carboxylation of purified PIVKA-II to prothrombin was detected by means of the one-stage coagulation assay and with 14CO2 incorporation. The reaction was dependent upon microsomal supernatant, vitamin K, ATP, Mg++ ions, O2, pH and temperature. Inhibitors were: warfarin, 2.4-dinitrophenol and factor II. From our experiments we concluded that PIVKA-II can act as a precursor of prothrombin and that PIVKA-II can be converted into the normal clotting factor in an in vitro cell-free system. In contrast to the systems published by others, here the converting enzymes are added to separately purified precursor proteins, so the system provides a way to study the nature of this reaction in more detail.
The Production of Heart Attacks and Cerebral Stroke by Thromboxane A2 Mixtures and their Prevention with Eg626 (Phthalazinol)
Authors: Shimamoto T. , Kobayashi M. , Takahashi T. , Takashima Y. , Motomiya T. , Morooka S. , Numano F.
Published: May 2019

Thromboxane A2 (TXA2) has been implicated as the causative agent in variant angina, myocardial infarction, sudden cardiac death and stroke. In order to verify experimentally such hypothesis, the TXA2-mixture was produced by washed rabbit or human platelets stimulated by thrombin (3U for 1.2 × 109 platelets) and the dose of TXA2 was measured by rabbit aortic strips.

The intracoronary (by our aortic balloon technique) or intracarotid injection of TXA2-mixture successfully produced variant angina-like response, fatal or non-fatal myocardial infarction and cerebral infarction resp. in 102 rabbits. The responses were recorded by a movie, polygraphic method including ECG, EEG and histological technique. The effective doses ranged from 1~2 μg of TXA2 in terms of angiotensin II for the intracoronary and 3 to 6 μg for the intracarotid injection resp. The mixture, kept at 37°C for 5 min, lost TXA2 and remained without effect, despite the presence of other vasocontrictors, showing the leading role of TXA2 for the occlusion of arterioles. The variant angina-like response, myocardial infarction and stroke were statistically significantly prevented by pretreatment of rabbits with therapeutic doses (0.1~1.0 mg/kg, i.v.) of EG626, an orally available potent TXA2-antagonistic and cAMPPDE-inhibiting substance, among drugs tested. The occlusion of arterioles by TXA2 by its time-matched vasoconstrictive activities after production of platelets aggregates as the plug was prevented by EG626 by reducing the grade of vasocontriction and platelet aggregation resulting into sliping away of the aggregates and the maintenance of the blood flow. Such facts may show the importance of EG626-type drugs in prevention of heart attacks and stroke as evidenced by its preliminary clinical results.
Patterns of Deposition of Blood Cells on the Luminal Surface of Jugular Veins Induced by Surgery or Infusion of Vasoactive Substances
Authors: Schaub R.G. , Stewart G.J.
Published: May 2019

The deposition of blood cells on canine jugular veins induced by intestinal anastomosis followed by a 4 hour post-operative interval or 4 hour intravenous infusion of vasoactive substances was studied by scanning and transmission electron microscopy (SEM, TEM). The vasoactive compounds infused were serotonin (200 ug/min), histamine (150 ug/min), or bradykinin (6 ug/min). Control dogs were infused with Tyrode’s solution alone. Three dogs were used in each group. At the end of the experimental period 90% of blood was removed by total body perfusion. Immediately after asystole the vessels were partially fixed in situ then removed for further processing. The pattern of cellular deposition varied with the experimental group. Control dogs exhibited the least response with only sparse deposition of individual leukocytes and erythrocytes. Anastomosis dogs exhibited the greatest response with extensive deposition of erythrocytes and leukocytes both a-lone and in association with fibrous material. The response of serotonin infused dogs was limited to deposition of patches of leukocytes with occasional erythrocytes. Both histamine and bradykinin infused dogs exhibited a sparse to moderate deposition of leukocytes and erythrocytes on most of the luminal surface. In addition, these animals also had extensive focal deposition of leukocytes and erythrocytes around valves and inflow areas of feeder vessels. The results suggest: (1) extensive deposition of thrombotic material can occur in the early post-operative period, (2) agents known to alter endothelial permeability can induce moderate general and extensive focal cellular deposition, and (3) the extensive focal deposition induced by histamine and bradykinin infusion was limited to stress areas.
Primary Structure of Plasminogen. Separation of two Lysine-Binding Domains and one Neoplasminogen (M.w. = 38,000)
Authors: Sottrup-Jensen L. , Petersen T.E. , Magnusson S.
Published: May 2019

The complete amino acid sequence of the heavy chain of human plasmin (residues 1-560) has been established. The complete sequence of the ‘light chain (residues 561-790) is accounted for but six overlaps remain to be established. We have located 17 of the 24 disulfide bridges. The remaining 7 can be assigned with high confidence on the basis of extensive homology of the light chain with serine proteases and of kringle domains K1-K5 (residues 83-161, 165-242, 255-333, 357-434 and 461-540, resp.) with the two kringle domains (residues 66-144 and 171-249) in bovine prothrombin. Of the two oligosaccharide groups one is glucosamine-based, attachedto Asn-288, the other galactosamine-based, attached to Thr-345. The former is either present or absent, the latter is heterogeneous. This variability could account for six or more of the observed electrophoretic variants of plasminogen. Degradation of native human plasminogen by porcine pancreatic elastase gave 14 small peptides (12 from the PAP region and two (residues 338-353 and 440-441, from between kringles 3 and 4 and 4 and 5, resp.), and three major fragments. Two of these, namely Kl+2+3 (residues 79-337/353 (M.w. 29,843. plus)) and K4 (residues 354-439 (M.w. 9,687)) each contain a lysine-binding site. The third,- neoplasminogen-Val-442 (residues 442-790) is activated faster than plasminogen (residues 1-790) with either urokinase or streptokinase indicating that the structure contained in residues 1-441 (PAP+K1-K4) is not necessary for activation of plasminogen, and raising quêtions regarding a possible role for leukocyte elastase in changing the affinity of plasminogen/plasmin for other proteins by removing lysine-binding sites. A 42-resi-due fragment (residues Asp-546 - Gln-575, -S-S-bridged to Lys-660 - Tyr-671) is a substrate for urokinase, which cleaves the same Arg-Val-bond (560-561) as in native plasminogen.
An Inventory of Plasminogen Activators in Human Plasma
Authors: Kluft C.
Published: May 2019

Plasma plasminogen (pro) activators were activated and precipitated completely in a euglobulin fraction by means of dextran sulphate. Total activator activity was measured by adding flufena-mate which rules out the influence of precipitated C1-inactivator on intrinsic activator system(s); extrinsic or vascular activator activity resistant to C1-inactivator was measured separately by adding extra C1-inactivator. In normal morning plasma, a total level of 100 + 15 (n=50) arbitrary blood activator units (BAU)/ml was detected; after 15 min venous occlusion, plasma contained 249 + 27 BAU/ml (n=7). The contribution of extrinsic activator amounted to a few percent in morning plasma and around 120 BAU/ml in occlusion plasma. The measured activator activity in morning plasma originates mainly from the intrinsic system(s) and was subdivided roughly into two equal components, one dependent on factor XII and one apparently independent of factor XII. The latter was found in Hageman trait (52 BAU/ml; n=3) and Fletcher trait plasma (44 BAU/ml; n=2). The factor XI I-dependent component was revealed by the correction of the deficiencies with factor XII (1%) and purified plasma kallikrein (100,000 and 300,000 daltons, 50-75%), respectively. Fitzgerald trait plasma (84 BAU/ml; n=1) did not show an altered response. Activator activity (10-15 BAU/ml plasma) found in partially purified kallikrein preparations of 100,000 as well as 300,000 daltons seems to be a property of the kallikrein. Thus, the total amount of activator activity found in morning plasma is made up by a few percent extrinsic activator, 10-15% kallikrein, 30-40% factor XI I-dependent activator and 40-50% unidentified component(s).
Inhibition of Platelet Reactivity by Endothelial Cells
Authors: Harker L.A. , Joy N. , Wall R.T. , Quadracci L. , Striker G.
Published: May 2019

The process of endothelial thromboresistance has been studied by measuring platelet reactivity to cultured human endothelial cells (EC), vascular smooth muscle cells (SMC), and fibroblasts (FB). Human C-serotonin labeled platelets in a concentration of 300,000 cells/ul and washed cultured cells (brought into monocellular suspension by EDTA buffer) were reacted in stirred plasma suspensions at 37°C while assessing light transmission, C-serotonin release and electron microscopy. SMC from adult arteries and veins induced aggregation and release (A-R) in a dose response fashion with maximal C-serotonin release of 62% ± 7 by 106 SMC/ml, whereas comparable reactivity was induced by 3 × 106 /ml umbilical vein SMC and 5 × 10 FB/ml. Trypsinization at 0.05% solution for 2 minutes destroyed the capacity of SMC and FB to induce A-R.

EC failed to induce aggregation and release in concentrations as high as 10 × 106 cells/ml. Moreover, the addition of EC to suspensions of SMC or FB in ratios as low as 1:5 prevented platelet A-R. The inhibitory effect of EC was not destroyed by trypsinization. Platelet A-R induced by ADP, epinephrine or collagen were similarly impaired by the addition of EC (106 cells/ml) while control studies using comparable numbers of SMC and FB produced no change. EC grown in the presence of indomethacin failed to inhibit platelet reactivity. ADP, epinephrine and collagen induced A-R were also significantly reduced by supernatant media from EC cultures. Supernatant media from SMC and FB failed to affect A-R. These results indicate that the lack of platelet reactivity to endothelial cells is mediated through endothelial cell synthesis of a soluble platelet prostaglandin inhibitor, probably prostacyclin.
Injury of Endothelial Cells in Culture Induced by Low Density Lipoproteins
Authors: Henriksen T. , Evensen S.A. , Carlander B.
Published: May 2019

The effect of low density lipoproteins on human endothelial cells in primary culture was investigated. The effect was evaluated by use of a Cr-release assay and phase contrast microscopy. When the cells were exposed to low density lipoproteins(LDL, d=l.019-1.063g/ml)suspended in RPMI 1640 the endothelial cells underwent extensive morphological changes and detached from the bottom of the culture vessel. Addition of lipoprotein-poor serum (d>1.21 g/ml) prevented the injurious effect of the LDL fraction at low concentrations, and to a lesser extent as the LDL concentration was raised. Unfractionated serum from hyperlipoproteinemic patients(type IIa) did not appear to injure endothelial cells. Addition of the LDL fraction, however, had a more pronounced effect on the cells when added to hyper-lipoproteinemic serum than to normal human serum. Thus, LDL as obtained by ultracentrifugation may have an injurious effect on cultured human endothelial cells. Secondly, hyperlipoproteinemic serum appears to contain factors which augment the effect of the added LDL fraction.
Simultaneous 51CR AND 14C-Serotonin Platelet Survival Measurements
Authors: Hanson S. , Harker L.A.
Published: May 2019

In vivo platelet reactions resulting in platelet utilization or release of constituents without platelet destruction have been studied in baboons by measuring isotopic disappearance of doubly labeled platelets using 51Cr as the cytoplasmic label to detect destruction and 14C-serotonin as a granular |abel to detect release. In 25 normal animals, the 14C-serotonin platelet survival (14C-SPS) was 6.0 days ± 0.2 compared with a Cr platelet survival (51Cr-PS) of 5.5 days ± 0.2, suggesting about 10% reutilization of the 14C label. Platelet labeling in vivo with 51C-serotonin showed that only 13% ± 2% of the injected activity became platelet bound; the remainder was cleared from the plasma within three hours. The platelets labeled in vivo survived normally (6.0 days ± 0.4). These results indicale that in vivo reutilization of serotonin is not extensive and that the process of labeling platelets with Cr in vitro does not appreciably shorten their survival in vivo. In other studies, animals with doubly labeled platelets received intravenous infusions of thrombin, ionophore R02-2985, arachidonic acid, ADP, collagen, plasmin, streptokinase, endotoxin, bovine factor VIII, and air bubbles. Thrombin or collagen infusions produced rapid, equivalent disappearance of both labels that correlated closely with decreases in platelet counts indicating that platelet destruction is not associated with reuptake of released C-serotonin by ambient circulating platelets. In contrast the infusions of R1I2-2985, plasmin or some studies with endotoxin induced selective disappearance of 51C-serotonin while 51Cr-PS was normal, reflecting platelet release in vivo without platelet destruction. With ADP, arachidonic acid, streptokinase and air bubbles platelets disappeared from the circulation transiently without loss of either label indicating transient platelet sequestration. We conclude that doubly labeled platelet survival studies are useful in characterizing platelet reactions and their pharmacologic alterations in vivo.
Platelet Survival and Platelet Serotonin Release in vivo in Various Animal Models
Authors: Cargill D.I. , Ryan R.J.
Published: May 2019

Decreased platelet survival is a clinically accepted indicator of increased platelet consumption, and in some cases may reveal “hyperthrombotic” states. Decreased platelet survival has also been demonstrated in the homocystinuric baboon and the hypercholesterolemic rhesus monkey (Ross and Harker). Treatment with antiplatelet drugs does increase platelet survival in patients as well as in experimental animals.

We have found platelet survival to be decreased in both uricemic and hypercholesterolemic guinea pigs.However, platelet survival in the atherosclerotic rabbit and the streptozotocin diabetic rat was normal. When atherosclerotic rabbits or diabetic rats were given platelets labelled with 51Cr and 14C-serotonin, they were found, five days after receiving the platelets, to have higher 51Cr/14C ratios than did their corresponding controls. This suggests a higher serotonin release from circulating platelets in these models.

The 51Cr/14C ratio in platelets may be a more sensitive measure of platelet activity in vivo than is platelet survival alone.
Defective Reticuloendothelial Clearance of Platelets after Whole Body Trauma
Authors: Kaplan John E. , Saba Thomas M.
Published: May 2019

Platelet microembolization has been suggested to result in microcirculatory and organ damage, especially pulmonary dysfunction, following trauma and shock. It is hypothesized that, in addition to increased platelet adhesiveness and aggregability, post-traumatic reticuloendothelial depression predisposes to microembolization by failure to clear altered platelets from the circulation. The presentstudy evaluated the short term (l hr) clearance and organ localization of radiolabelled homologous platelets in normal rats and rats following sublethal Noble-Collip drum trauma. After preparation of citrated platelet rich plasma from normal rats, platelets were collected and labelled with 51cr in citrated saline. Platelets were injected IV (2 mg protein/100 g BW) and radioactivity determined in serial 100 ul blood samples and in the liver, lung and spleen at 60 min. Platelet disappearance conformed to a 2 compartment exponential clearance. Velocity of clearance in the most rapid compartment correlated (r=0.93) with hepatic platelet localization while velocity of the second compartment correlated (r=.91) with splenic platelet localization. Clearance rate of the rapid compartment was depressed at 1 hr after trauma (k=0.08l min-1) compared to control animals (k=0.29 min-1) and was elevated at 24 hr (k=0.52 min-1). These changes were associated with a 35% decrease in hepatic platelet localization at 1 hr and a 55% increase above normal at 24 hr. Splenic platelet localization was decreased 31% by 3 hr following trauma. Pulmonary platelet localization was increased at all times following trauma with a maximal 320% increase 3 hr post-trauma. It is concluded that the post-trauma state is associated with a defect in the RES clearance of altered platelets which may in turn augment embolization of platelets in the microcirculation.
The Role Of Fc Fragment Receptor in Immune Injury of Human Platelets Induced by Protein a of Staphylococci
Authors: Hawiger J. , Steckley S. , Hammond D. , Cheng C. , Tjmmons S. , Prez Des R. M.
Published: May 2019

The ability of the Fc fragment to trigger an immune injury to human platelets challenged with immune complexes or aggregated IgG was mainly observed in plasma-free medium. We now report immune injury to human platelets in autologous plasma when Protein A-bearing staphylococci were added to the system. Protein A, a cell wall constituent of pathogenic staphylococci interacts with the Fc fragment of IgG making it reactive with the membrane receptor. Aggregation amd release of [3H]serotonin from human platelets suspended in autologous plasma occured when at least two Protein A-bearing staphylococci were added per one platelet. Partial stripping of Protein A by trypsinization reduced the extent of platelet injury. When Protein A was solubilized and isolated by affinity chromatography, release of [3H]serotonin by soluble Protein A-IgG complexes occured only in plasma-free medium. Using radiolabelled Protein A, we demonstrated that release of [3H]serotonin was paralleled by the binding of [125I]Protein A-IgG complexes to platelet membrane. This binding was dependent on the Fc fragment and its receptor since the removal of the Fc fragment abolished the binding of Protein A to platelets.Thus, immune injury to human platelets has been demonstrated in two systems: (1) when Protein A was in solid phase, i.e., attached to staphylococci, platelet injury by Protein A-IgG occured in plasma, and (2) when Protein A was in fluid phase, i.e., isolated from staphylococci, injury occured only in plasma-free medium. Both mechanisms required the Fc fragment of IgG to interact with the membrane receptor on human platelets.
Complement-Dependent Action of HLA-Specific Antibodies on Human Platelets
Authors: Heinrich D. , Mueller-Eckhardt C. , Bitter-Suermann D.
Published: May 2019

HLA-antibody-induced platelet alterations are mediated by (a) complement-independent (Heinrich et al, Brit. J. Haematol., 35, 443, 1977) and (b) complement-dependent mechanisms. To prove the latter hypothesis platelet-rich plasma (PRP) of HLA-typed donors was incubated with HLA-specific sera and various inhibitors of platelet function (prostaglandin-E1 (PGE1), acetyl-salicylic acid (ASA), N-ethyl maleimide (NEM), adenosin, 2-desoxy-glucose + antimycin A (2-DOG + ANT.A)), furthermore an inhibitor of complement activation (cobra venom factor (CVF)) and EDTA or heparin known to interfer with platelet function and complement activation. Methods: Platelet aggregation (Born), Hc-seroto-nin release and 51Cr release. Results: Contrary to collagen-and thrombin-induced platelet alteration, PGE1, NEM, ASA, adenosin and 2-DOG+ANT. A exhibited only moderate inhibition of antibody-induced platelet alteration. However, EDTA, heparin and CVF+PGE1 strongly inhibited antibody-induced platelet alteration. The results support the concept of two pathways for specific HLA-antibody action on platelets: (a) a complement-independent mechanism resulting in aggregation and release and (b) a complement dependent, lytic mechanism.
Immunological Abnormalities in Childhood Chronic Idiopathic Thrombocytopenic Purpura (ITP)
Authors: Stuart Marie J. , Tomar Russell H. , Miller Merrill L. , Davey Fredrick R.
Published: May 2019

Chronic ITP, unlike acute ITP, may be the result of an underlying immunologic disorder. To examine this possibility 3 children, and their families (totalling 13 members) were studied in an attempt to identify underlying abnormalities in cellular and humoral immunity. All 3 patients had their disease for more than 1 year. All had shortened platelet life spans. None were receiving drugs, and none had had their spleens removed. In these patients, 2 of 3 were found to have decreased numbers of T lymphocytes and PHA reactivity; 2 of 3 had dysgammaglobulinemias; 1 of 3 had decreased isohemagglutinins, and all 3 had abnormal reactivity to a variety of intradermal antigens. In the family members these same abnormalities were found with increased frequency. 6/13 were found to have decreased numbers of T lymphocytes and PHA reactivity; 6/13 dysgammaglobulinemias and 1/13 decreased isohemagglutinins. In addition, autoantibodies such as a biologically false positive serological test for syphilis, and antithyroid antibodies were also found. A parent of one of the propositi, and 2 asymptomatic siblings of another were also documented to have shortened platelet life spans in the presence of normal platelet counts. The HLA antigens, A3 and B7 were found in all 3 families. These 2 HLA antigens have previously been documented to occur with increased frequency in a variety of “auto-immune” disorders. These studies demonstrate that chronic ITP occurs in families with immunologic defects, and suggests that this disease has a genetic rather than an exclusively environmental basis.
Cell Proliferation and DNA Synthesis in Organizing White Mural Non-Occlusive Aortic Thrombus in Rabbits
Authors: Rabinovitch J. , Weigensberg B.I. , More R.H. , Gotlieb A.
Published: May 2019

White mural non-occlusive thrombus induced experimentally in the aortae of rabbits contains appreciable numbers of proliferating cells which contribute bulk and mass to the lesion. As these cells proliferate they produce appreciable amounts of collagen, elastin, and mucopolysaccharide which make the lesions resistant to dissolution by enzymes. In this study the proliferation of cells was monitored at serial time intervals up to 60 weeks by radioautography and chemical measurement of [H3]thymidine incorporation into DNA in order to determine over what time intervals appropriate antiproliferative drugs might be tested to arrest the growth of these cells. DPM [H3 thymidine incorporation into DNA became significant on Day 2, remained very high until Day 14 and then declined. The concentration of DNA per mg delipidated dry weight of thrombus between Day 0 and 4 was highly variable (6 to 60 μg/mg) due to the highly variable ratio of nucleated cells (monocytes and polymorphonuclear leukocytes) to fibrin and platelets. Between Day 0 and 4 weeks the DNA concentration became less variable as the polymorphsand monocytes were replaced by macrophages and spindle-shaped cells (10 to 20 yg/DNA per mg delipidated lesion). After 4 weeks DNA concentration declined continuously until by 60 weeks there was 2 ± 1 μg DNA/mg delipidated dry weight of lesion as it was diluted by collagen, elastin, and mucopolysaccharide. It is concluded that if antiproliferative drugs are to be used, they must be started as early as Day 2 and be maintained for at least 2 weeks.
The Platelet Strip: A New Model for the Study of the Mechanochemical Properties of a Platelet Aggregate
Authors: Salganicoff Leon , Russo Matteo , Loughnane Michael
Published: May 2019

A new model, whose purpose is the quantitative study of the corelation between the force of contraction of a platelet aggregate and morphological, biochemical or pharmacological parameter is described. This preparation is made by centrifuging cold recalcified PRP over a sheer nylon, elastic mesh kept stretched in a hoop inside a specially made centrifuge tube with a solid flat bottom. As a result, the mesh becomes impaled in the flat platelet pellet giving it structural support. The supernatant PPP is decanted and the pellet washed with cold Tyrodes solution to eliminate as much residual plasma as possible. Reheating the preparation at 37° activates the aggregation of the platelets and the coagulation of the residual fibrinogen. The final result is a disc of approximately .3mm thickness with a density 7-8 × 107 plat./mm3. The disc is disassembled, cut in strips which are hung using classical techniques of vascular smooth muscle in a bath, where the force produced by its contraction can be measured either isotonically or isometrically. Forces up to 500-700 mg per strip are produced at maximal contraction. The behavior of the preparation can be followed for periods of up to 5 hours. Under the effect of drugs it shows reversible contraction-relaxation cycles in an all or none mode. Kcontraction for epinephrine was 10-8M; nor-epinephrine, 10-7M. Krelaxtaction for PGE1 was 10-9M: sodium nitroprusside, 10-8M: (+)propranolol, 10-6M; phentolamine, 10-4M. It also shows reversible, ultraviolet-induced photorelaxation when excited at 366 nm. Similarities with aortic vascular smooth muscle and morphological characteristics will be discussed.