bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathogenic Role Of Thrombus Formation In The Microcirculation Of Mucosa In The Gastrointestinal Defence Mechanism
Authors: Kondo M , Yoshikawa T , Furukawa Y , Takemura S , Fukumoto K
Published: July 2018

There is no doubt that impaired blood supply to the gastrointestinal (GI) tract causes various pathological changes in the digestive mucosa by disrupting the defence mechanism. Ischemic change of the intestinal mucosa is classified into (1) occlusive mesenteric ischemia and (2) non-occlusive mesenteric ischemia, and intravascular clot formation has been investigated mainly in the former category.

However, GI-bleeding of unknown origin is often experienced during the management of patients in disseminated intravascular coagulation (DIC), while to which neither the mechanism nor the specific treatment has been indicated. In the present study, the role of the microcirculation in the GI- defence mechanism is investigated from the standpoint of DIC.

Single injection of E. coli endotoxin to dogs resulted in hemorrhagic necrosis of the GI mucosa within 8 hrs, and this was prominent in the small intestine where hemorrhagic change started at the top of villi. Microscopic examination showed tortuous thrombus formation in the microcirculation of the mucosa.

When the jejunal mucosa was treated directly by protease inhibitors such as tranexamic acid or aprotinin prior to endotoxin, hemorrhagic necrosis of the area was successfully prevented, suggesting that proteolytic enzymes either in the GI-cavity or in the tissue digested the ischemic mucosa.

Systemic pretreatment of rats with heparin was effective in preventing the hemorrhagic necrosis, strongly indicated that blood supply to the microcirculation of the GI-mucosa played a role in the GI-defence mechanism.

It is concluded that in the managing patients with DIC, one should be careful to protect GI-bleeding which often appear in the form of hemorrhagic necrosis. Besides the use of systemic anticoagulants, direct contact of protease inhibitors with GI-mucosa might be of help in the protection or the treatment of GI-bleeding in DIC.
Effect Of Neuraminidase On Plasmin Formation By Urokinase And The Vascular Plasminogen Activator
Authors: Geiger M , Binder B R
Published: July 2018

It was the aim of the present study to determine whether sialic acid residues in the activator or substrate molecule play a role in the activation of plaminogen to plasmin by the vascular plasminogen activator (VPA) and urokinase (UK).

Using a purified test system with UK (Mr=31.000, human urine), VPA (Mr=70.000, cadaver vessel eluates) as activators, Glu-plasminogen as substrate, and pretreatment with neuraminidase (C.perfringens,1.51U/mg) it could be shown by quantification of the formed plasmin with S-2251 as substrate that neuraminidase treatment enhances plasmin formation by UK as well as by VPA. Using Sepharose-bound neuraminidase (C.perfringens, 20-30U/g Sepharose) it could be shown that neuraminidase affects the substrate in a dose and time dependent fashion, while the activity of the activators seems to remain unchanged. Treatment of plasminogen with neuraminidase in a concentration of 2.5 mU per μmole plasminogen for 90 min. at 37°C increased the plasmin formation by VPA by about 50% which was the same as for UK.

Kinetic analysis of plasmin formation from neuraminidase treated plasminogen by UK and VPA suggests increased binding of the substrate to the activators (unchanged Vmax and decreased KM). The enhancing effect of fibrin on plasmin formation by VPA seems to remain unaltered by neuraminidase treatment of the plasminogen.

These data suggest that, as in the case of other protease substrates, removal of sialic acid residues results in increased cleavage of the substrate, i.e. enhanced plasminogen activation, regardless of the type of activator.
Further Characterization Of SK-Potentiator Of Plasminogen
Authors: Takada A , Mochizuki K , Takada Y
Published: July 2018

Streptokinase (SK) forms a complex with human plasminogen (plg) or plasmin, and the resulting complex (SK-activator) functions to convert plg to plasmin. We have indicated that human plasma contains a factor (SK-potentiator) which potentiates the capacity of SK to activate human plg. SK-potentiator has a molecular weight of 240,000, and composed of β and γ-chains of fibrinogen, α-chain being degraded. SK-potentiator crossreacts with anti-FDP-Y fragment. Immunodiffusion shows that SK-potentiator has an antigenic determinant in common with both FDP-Y and fibrinogen, and the other determinant not in common with fibrinogen but FDP-Y. Early FgDP potentiates SK-activator activity as much as SK-potentiator, but further degraded FgDP potentiates less than fibrinogen which still enhances SK-activator activity. The addition of thrombin to FgDP or SK-potentiator enhances SK-activator activity more than SK-potentiator. Thus removal of fibrinopeptides from FgDP or SK-potentiator results in better potentiator activity. When tranexamic acid (l mM) was added to the mixture of Glu-plg and UK, the activation of Glu-plg was enhanced, but tranexamic acid (l mM) added to SK-activator caused a decrease in SK-activator activity. The addition of fibrinogen or SK-potentiator to the mixture of tranexamic acid and SK-activator prevented the decrease of SK-activator activity to some extent, which may indicate that SK-potentiator competes with tranexamic acid for lysine binding sites (LBS) of plg and SK-potentiator forms a complex with SK-activator in spite of the presence of tranexamic acid. It is proposed that SK-potentiator binds with LBS of plg part of SK-activator and SK combines with light chain part of plg, the resulting SK-plg-potentiator complex being the better activator than SK-plg or SK-plasmin complex.
Plasminogen Activator (PA) Activity Of Urokinase (UK) , The Vascular Plasminogen Activator (VPA) , Plasma Kallikrein (PK) , And Hageman Factor (HF) In The Presence And Absence Of Fibrin
Authors: Binder B R , Beckmann R , Jörg M
Published: July 2018

It could be shown previously by us and others that the PA activity of purified VPA is increased in the presence of fibrin, while the activity of UK remains unchanged. However, there had been some discussion on the mechanisms involved and the relative potency of the different PAs in man. In the present study kinetic analysis of plasminogen activation with different PAs was performed in the presence and absence of fibrin.

Human UK (urine, Mr=56.000) , VPA (cadaver vessel eluates) , PK (HF-fragments activated) were purified to apparent homogeneity, and active sites were determined by 3H-DFP incorporation. Plasminogen activation was evaluated by incubation of different amounts of PAs with Glu-plasminogen at different substrate concentrations with and without fibrin (fibrinogen- Sepharose treated with thrombin) at 37°C for 3 to 120 minutes; the plasmin generated was measured by a synthetic para- nitroanilide substrate.

Under these conditions VPA exhibited an apparent KM≃0.5 μM and a kcat≃0.15 s-1. In the presence of fibrin KM decreased significantely (<<0.01 μM) while kcat remained unchanged; untreated fibrinogen-Sepharose had no effect. UK, on the other hand, showed an apparent KM≃1 μM and a kcat≃0.5 s-1 which were both unaffected by fibrin. PK cleaved plasminogen with an apparent KM≃0.2 μM and a kcat≃0.004 s-1 regardless of the presence of fibrin. Preliminary data using intact HF together with kaolin and phospholipids revealed an apparent KM for plasminogen activation of that mixture of 3.5 μM and a kcat≃0.07 s-1 which were again unaffected by fibrin.

From these data it can be concluded that in the absence of fibrin UK is a more effective PA than the others, while in the presence of fibrin VPA is by fare the most effective one. From kinetic analysis PK and HF should play only a minor role in plasminogen activation.
On The Fibrinolytic Properties Of Single-Chain And Two- Chain Human Tissue Plasminogen Activator
Authors: Rijken D C , Hoylaerts M , Collen D
Published: July 2018

Tissue plasminogen activator from pig hearts may be isolated as a single-chain or as a two-chain molecule (Wallén et al., 1980). The present report deals with the two molecular forms of a plasminogen activator (tissue plasminogen activator-like) secreted by human melanoma cells in culture. A single-chain form was prepared by addition of aprotinin to the culture media and during the purification procedure while a two-chain form or a mixture of both was obtained in the absence of aprotinin.

The fibrinolytic activities of the two forms were comparable on fibrin plates and in a clot lysis time system. Analysis of the molecular structure of 125I-labeled plasminogen activator by dodecyl-sulfate-gel electrophoresis, revealed that the single-chain form was converted into a two- chain form during the lysis of fibrin. The plasminogen activating properties of the two molecular forms were therefore measured in the presence of aprotinin, which prevents the conversion. Aprotinin (1,000 KIU/ml) was incorporated in a fibrin clot (1 mg/ml) containing plasminogen activator (20 ng/ml), 125I-labeled Glu-plasminogen and varying amounts of unlabeled Glu-plasminogen. The amount of plasmin formed after 30 min was quantitated by measuring the radioactivity migrating in the position of the plasmin B-chain on dodecyl-sulfate-gel electrophoresis under reducing conditions. The rate of plasmin formation obeyed Michaelis-Menten kinetics with Km = 2.5 μM and kcat = 0.6 s-1 for the single-chain form and Km = 1.1 μM and kcat = 0.3 s-1 for the two-chain form.

Although the conversion of the single-chain tissue plasminogen activator into a two-chain form during fibrinolysis might have a regulatory function, these kinetic parameters of the plasminogen activation do not support this hypothesis.
Three Dimensional Computer Graphics Models Of Bovine Factor Xa, Factor IXa And Thrombin
Authors: Furie B , Bing D H , Furie B C , Robison D J , Burnier J P , Feldmann R J
Published: July 2018

Three dimensional structural models of the heavy chain of Factor Xa, the heavy chain of Factor IXa, and the B chain of thrombin have been developed using a computer molecular graphics display system. These models are based upon the sequence homology of these proteins with chymotrypsin and trypsin and the assumption that the three dimensional structures of the peptide backbones of these proteins are nearly identical to the known backbone structure of trypsin and chymotrypsin. Sequence alignments of each protein with the digestive proteases were based upon regions of sequence homology, location of disulfide bonds, and preservation of β-barrels. Regions of insertion and deletion relative to the chymotrypsin backbone were identified and incorporated into the backbone structure. Factor Xa (heavy chain) and Factor IXa (heavy chain) contain 5 insertions and 4 deletions. Thrombin (B chain) contains nine insertions and two deletions. Factor Xa, Factor IXa, and thrombin models were generated by substituting their respective amino acid sequences into the model of the peptide backbone. The molecular surfaces of these models each suggest unique topographical and physicochemical properties. In-contrast, the core of these proteins are nearly identical. One area of the molecular surface, the active site, is also highly conserved despite marked differences in substrate specificity among these enzymes. The extended substrate binding regions, surrounding the active site, are highly substituted and also contain areas of both insertions and deletions. These models predict that the functional differences that distinguish these serine proteases are manifestations of the molecular surface differences. It would appear that substrate recognition and specificity determinants of Factor Xa, Factor IXa, and thrombin reside in an extensive surface surrounding the active site, but not exclusively within the active site.
Binding To Phospholipid Protects Factor VIII From Inhibition By Human Antibodies
Authors: Barrowcliffe T W , Gray E , Kemball-Cook G
Published: July 2018

Previous studies with activated Factor IX concentrates have suggested that they may contain a form of Factor VIII clotting activity (VIII:C) which is partly protected from inactivation by antibodies. A possible mechanism for such protection is binding to phospholipid. The interaction between Factor VIII, phospholipid and human antibodies to Factor VIII was studied by a two-stage clotting assay, and by a fluid-phase immunoradiometric assay for Factor VIII clotting antigen (VIII C:Ag).

In the two-stage thrombin generation assay, Factor VIII:C was rapidly destroyed by human antibodies, even in the presence of optimal phospholipid. However, preincubation of Factor VIII with phospholipid before addition of antibody protected the Factor VIII from inactivation, resulting in the production of much more thrombin.

In assays of VIII C:Ag, pre-incubation of Factor VIII with phospholipid before addition of labelled antibody reduced the amount of detectable antigen. The reduction was greater with increasing phospholipid concentration, up to 60% of the original antigen being ‘lost’ at a total phospholipid concentration of around 250 μg/i.u.

These results suggest that human antibodies to Factor VIII are directed largely at its phospholipid binding site. The protection of Factor VIII from inactivation by complexing with phospholipid could have important clinical applications in treatment of haemophiliacs with inhibitors.
A New Clinical Preparation Of High-Purity Porcine Factor VIII:C Concentrate
Authors: Costello W P , Middleton S M , Malia R G
Published: July 2018

A polyelectrolyte-fractionated porcine Factor VIII concentrate (Hyate:C, Speywood Laboratories, average specific activity: 10 U Factor VIII:C/mg, platelet aggregating factor (PAF): 0.25 U/ml) has been recently available. Clinical use of this product for the treatment of patients with antibody to Factor VIII (13 haemophilic and 1 non- haemophilic patients for a total of 26 courses of treatment) has shown that: 1/ multiple courses of therapy can be given with both good clinical tolerance and good clinical efficacy, 2/ with one exception, no patient developed thrombo- cytopaenia and 3/ with the exception of one case, in which the patient has been treated with human Factor VIII prior to the infusion of porcine Factor VIII. the expected anamnestic response did not occur. These clinical findings prompted us to further purify the porcine Factor VIII concentrate for clinical use.

Cryoprecipitate, prepared from frozen porcine plasma, is dissolved in low ionic strength Tris buffer at pH 6.8 and adsorbed with aluminium hydroxide gel. The ionic strength is then adjusted to 0.3 M NaCl before loading on a column of charged and equilibrated polyelectrolyte E-5. After washing the column with 4 bed volumes of buffer, the proteins were eluted with 1 M NaCl at pH 6.5 and precipitated by 20 % polyethylene glycol 4000. The redissolved precipitate is then chromatographed on a preparative column of Sepharose CL-4B. The void volume is sterile-filtered and lyophilised. The new preparation has a specific activity of approximately 100 U Factor VIII:C/mg with a PAF level of 3.3 U/ml. This represents an improvement of 10 fold in specific activity over the existing concentrate.

It is expected that the new concentrate will also show low immunogenicity which can only be revealed by clinical results. A clinical trial is envisaged within the next two months.
Postoperative Platelet Accumulation In The Lungs: Can It Be Measured In Surgical Patients And Does It Matter?
Authors: McCollum C N , Hawker R J , Norcott H C , Hail C , Drolc Z , Miller M R
Published: July 2018

Pulmonary microembolisation as a result of trauma, sepsis or major surgery may be an aetiology for shock lung. The postoperative kinetics of 111-Indium labeled autologous platelets have been studied to determine whether they accumulate in the lungs, and whether this relates to subsequent pulmonary function.

One day prior to major surgery platelets from 10 patients were labelled with 111-Indium-oxine and reinjected. Isotope emissions were counted over the lungs and aortic arch preoperatively, 2.5 hours following surgery and daily for 7 days. Screen filtration pressure (SFP) measurements of aggregates in femoral vein blood was estimated 30 minutes and 3 hours postoperation. Arterial PO2 was measured preoperation and on days 1 and 7 following surgery.

The ratio of isotope emissions lung/aortic arch increased following surgery indicating pulmonary accumulation of platelets. The preoperative ratio (mean ± SEM) of 0.38 ± 0.017 rose to 0.47 ± 0.039 (p<0.05) 2.5 hours following surgery and to 0.56 ± 0.052 (p<0.01) the following day. This ratio had returned to preoperative levels by day 4. Immediate postoperative platelet accumulation in the lung (the rise in lung/ aortic arch ratio) correlated closely with SFP measurements (r 0.89, p<0.01) and the fall in arterial PO2 one week following surgery (r 0.69, p<0.05).

These results indicate that circulating platelets aggregate during surgical shock and then accumulate in the lungs. This process is associated with postoperative hypoxia.
Pulmonary Embolism: Trends In Edinburgh Surgical Units Over Twenty Years
Authors: Ruckley C V
Published: July 2018

The annual statistical records (audit) of ten general surgical units for the years 1959, 1964, 1969, 1974 and 1979 have been analysed to find the trends in incidence of pulmonary embolism in patients who died - with particular reference to patients normally expected to have a good prognosis.

The operations for the 5 years totalled 59,880. There were 2,593 deaths and 1,181 autopsies (46%). The total annual operations rose from 9,757 in 1959 to 13,830 in 1979 while the death rate fell from 6% to 4%.

Pulmonary embolism proven at autopsy (150) plus clinical diagnosis when autopsy was not performed (41) was reported in 191. There was a fall in incidence from 0.4% of operations in 1959 to 0.2% in 1979; but the validity of this was obscured by a fall in autopsy rate from 59% to 34% over the same period. However in patients below the age of 80 who should have had a good prognosis (i.e. did not have metastatic malignancy or end-stage cardiorespiratory disease) the autopsy rate remained relatively constant at a mean of 76% throughout the period. In this important group the incidence of pulmonary embolism fell progressively from 0.27% in 1959 to 0.06% in 1979.

In general surgical patients who would normally be expected to have a good prognosis the risk of fatal pulmonary embolism has steadily diminished over the past twenty years, although prophylactic methods have only been in general use for the past 5 years.
Deep Vein Thrombosis During Oral Contraception. An Analysis Of 57 Patients
Authors: Bergqvist A , Bergqvist D , Hedner U
Published: July 2018

A phlebographic and haemostatic analysis has been made of 57 patients developing deep vein thrombosis while on combined oral contraceptives containing 30 or 50 μg estrogen. The diagnosis was based on ascending phlebography. One patient had bilateral, 18 rightsided and 38 leftsided thrombosis. The leftsided dominance was seen only in patients taking 50 μg estrogen daily. The thrombotic process was significantly more extensive and more proximally located in patients with leftsided thrombosis. Thirty-three percent of the rightsided and 8% of the leftsided thrombi embolised, a difference which is significant. Six to twelve months after the acute thromboembolic episode an analysis was made of platelet function, the coagulation and fibrinolytic systems. Only 5/57 patients had an excessive increase of f. VIII:C (>250%) and none had low AT III levels. Thirty-two percent of the patients had a defect in their vessel wall fibrinolysis (a defective release capacity and/or a decreased fibrinolytic activity within the vessel wall) and these patients developed rightsided thrombosis significantly more often.

Judging from our findings a defective vessel wall fibrinolysis seems to be of pathogenetic significance for the development of deep vein thrombosis especially on the right side during oral contraceptive medication. The more extensive leftsided thrombi seemed to be dependent on the estrogen dose and not to the same extent associated with an impaired vessel wall fibrinolysis.
Effects Of Bay g 6575 On Platelets And On Vascular PGI2 Production
Authors: MacIntyre D E , Salzman E W
Published: July 2018

Bay g 6575 (1-[2-(β-naphthyloxy) ethyl]-3-methy1-2-pyrazolin-5-one) exerts a protective effect in several animal models of thrombosis. To elucidate its mechanism of action, we examined the effects of Bay g 6575 on platelets and on vascular PGI2 production. In vitro addition of Bay g 6575 (200 μM) to human citrated platelet rich plasma (PRP) did not inhibit aggregation induced by ADP or U44069, or augment inhibition of ADP-induced aggregation by PGD2, PGE1, PGI2 or papaverine. When added to isolated human or rat vascular rings, Bay g 6575 (200 μM) did not stimulate production of PGI2 or 6-oxo-PGF1α. Ex vivo studies one hour after administration of Baya g 6575 to rabbits (10 mg/kg, i.a.) or rats (100 mg/kg, p.o.) revealed no inhibition of ADP-induced aggregation or enhancement of the level of “circulating” PGI2 as measured by bio-immunoassay. When production of anti-aggregating activity by vascular rings from Bay g 6575 treated (B) and Control (C) rats were compared, in 6 of 8 experiments B inhibited more than C and B produced more 6-oxo-PGF1α than C (mean increase in B ± s.d.=74.3 ± 35.7%, range 42-135%). Production of antiaggregatory activity by “exhausted” C rings was enhanced by B>C platelet free plasma. In all cases, the inhibitor of aggregation produced by B and C rings acted on both human and rat PRP, and its effects could be reversed by anti-PGI1 antibodies that neutralize PGI2>6-oxo-PGE1>PGD2. When exogenous PGI2 was incubated with (exhausted) aspirin treated vascular rings, the duration of action of PGI2 was longer in the presence of B rings than C rings.

Bay g 6575 has no direct effects on platelets or on vascular tissue. Its antithrombotic activity appears to be caused by regulation of PGI2 synthesis and metabolism, an effect mediated by factors, possibly Bay g 6575 metabolites, present in plasma after in vivo administration.
Altered Factor VIII Antigen: A Sensitive Indicator Of Endothelial Damage
Authors: Carvalho A C A , Bellman S , Saullo J , Lauer J , Zapol W M
Published: July 2018

Acute respiratory distress syndrome (ARDS) is characterized by pulmonary vascular injury and altered endothelial permeability. Since Factor VIII antigen (VIII:Ag) is synthesized by endothelial cells, we studied FVIII:Ag and coagulant (VIII:C) activities in 100 patients with ARDS. Factor VIII:C was determined by one-stage assay, FVIII:Ag by quantitative immunoelectrophoresis and FVIII complexes by cross-immunoelectrophoresis (CIE). We studied 56 patients with mild acute lung injury (lobar infiltrate, mortality 30%), 30 with moderate ARDS (mortality 60%), and 14 with severe ARDS (mortality 90%) secondary to various etiologic agents. The results are expressed in % (Mean±SEM) as shown below: In all ARDS groups, there was a marked increase in FVIII: Ag by 4- to 5-fold the normal average. By CIE, both slow- and fast-moving components of FVIII:Ag displayed abnormal patterns. In moderate and severe ARDS, there was an increase in the fast-moving component of FVIII:Ag, mimicking vonWillebrand’s defect; this FVIII change may result in hemorrhage in the lung and elsewhere. In contrast, in mild ARDS there was an increase in FVIII:Ag slow-moving component which may account in part for the increase in pulmonary micro and/or macrothrombi formation. Recovery from ARDS resulted in return towards normal of FVIII:Ag patterns. Thus, FVIII:Ag appears to be a sensitive indicator of pulmonary endothelial injury and repair. In addition, our data strongly suggests that excessive concentrations and altered FVIII:Ag patterns may contribute to the thrombo- hemorrhagic complications commonly seen in acute lung injury.
Additional Glycoprotein Defects In Glanzmann’s Thrombasthenia Platelets
Authors: McGregor J L , Clemetson K J , James E , Capitanio A , Dechavanne M , Lüscher E F
Published: July 2018

Glanzmann’s thrombasthenia (G.T.) platelets are deficient in 2 major membrane GP (IIb and IIIa). In order to investigate if these are the only defects in this disorder, platelets from G.T. patients and from healthy donors were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetylgalactosamine residues). Labelled or unlabelled platelets were solubilized in sodium dodecyl sulphate (SDS) and separated by 2-dimensional polyacrylamide gel electrophoresis, first according to isoelectric point and then according to molecular weight. Glycoproteins from unlabelled platelets separated by 2-dimensional electrophoresis were identified by binding of 125I-labelled Lens culinaris lectin (mannose, glucose specific) GPIIbA1 and IIIaA1 were absent in one G.T. patient while in others lower amounts of 2 GP were found in positions similar to these GP. Major membrane GP (IbA1, IbA2, IbB1 and IIIbA1) had more intensely labelled terminal sialic acid moieties in G.T. platelets than in normals. A major membrane GP designated Ic had an altered pi and its penultimate galactose/N-acetyl galactosamine residues labelled more intensely in G.T. platelets than in controls. One high M.Wt. GP and a number of lower M.Wt. GP (IVa, IVb and VII) normally found in platelets of healthy donors were absent in G.T. platelets. These results indicate strongly that there is a major perturbation of the platelet surface in G.T.
The Reduced Aggregation Response Of Bernard-Soulier Platelets To Thrombin May Be Related To An Abnormal Glycoprotein V
Authors: Nurden A T , Dupuis D
Published: July 2018

Both platelet membrane GP Ib and GP V have been proposed as receptors for the activation of human platelets by thrombin. Bernard-Soulier (B-S) platelets exhibit a reduced aggregation response to thrombin with a lag phase that precedes aggregation. When B-S platelets, whose surface proteins had been labelled with (125I), were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by PAS-staining, Coomassie blue staining or autoradiography, the apparent absence of GP Ib and the normal presence of GP IIb, IIIa and IIIb was demonstrated. On the basis of such studies several authors have stated that “GP I” is the thrombin receptor. However, GP V is not located by the above procedure, requiring more sensitive analytical methods for its detection. To meet this requirement washed platelets isolated from 3 B-S patients have been treated sequentially with neuraminidase, galactose oxidase and sodium(3H,)-boro- hydride. The labelled platelets were analysed by SDS-PAGE using 7-12% gradient acrylamide gels and the (3H,)-labelled GP’s located by fluorography. In addition to the GP Ib defect the platelets of each B-S patient were lacking the band corresponding to GP V of normal platelets. In agreement with previous studies we observed that when (3H,)-labelled normal human platelets were incubated with thrombin GP V (Mr=82,000) was hydrolysed,and that this was accompanied by the appearance of a labelled glycopeptide (Mr=69,500) in the supernatant. When (3H)-labelled B-S platelets were treated with thrombin no labelled glycopeptide was located. GP V could therefore be either absetit from B-S platelets or have a modified carbohydrate composition rendering it insensitive to the analytical procedure used. Interpretations into the reduced aggregation response of B-S platelets to thrombin should be extended to include a possible GP V defect.
Tryptic Peptide Maps Of The Major Human Blood Platelet Membrane Glycoproteins, Ia, IIa, IIb, IIIa, IIIb and IIIc
Authors: McGregor J L , Clemetson K J , James E , Clezardin P , Dechavanne M , Lüscher E F
Published: July 2018

Some major platelet membrane glycoproteins separated by 2-dimensional polyacrylamide gel electrophoresis (isoelectric focusing, discontinuous SDS-gel electrophoresis) have been characterized by 2-dimensional tryptic peptide mapping. Human platelets were isolated, washed and surface-labelled by lactoperoxidase-catalyzed iodination. Labelled platelets were solubilized in SDS and separated by 2-dimensional gel electrophoresis under non-reducing conditions in the 1st dimension and either reducing or non-reducing conditions in the 2nd dimension. Alternatively, labelled platelets were solubilized in sodium deoxy- cholate (1 %) and the glycosylated components were isolated by lectin affinity-chromatography on Lens culinaris lectin or concanavalin A lectin and separated by 2-dimensional gel electrophoresis. The glycoproteins were cut out from 6 % polyacrylamide gels, after being identified with Coomassie blue staining and indirect autoradiography by their pI and molecular weight. Tryptic maps were prepared according to the method of Elder et al. The tryptic maps of GPIa, IIa, IIb, IIIa, IIIb and IIIc are different, with each showing a characteristic pattern with the possible exception of Ia and IIb which showed certain similarities in both the reduced and non-reduced states. GPIIa which is clearly separated under non-reducing conditions, appears from its tryptic map to be present in the Ib region when reduced. Thus this technique clearly identifies each GP by a parameter in addition to pI and molecular weight.
Adenosine 5’-O-(2-Thiodiphosphate) (ADP-β-S) Is A Partial Agonist At The ADP Receptor Of Human Platelets
Authors: Cusack N J , Hourani S M O
Published: July 2018

ADP induces human platelet aggregation and inhibits the stimulation of platelet adenylate cyclase by prostaglandin E1 (PGE1), but analogues of ADP in which the diphosphate group is modified retain only weak aggregating activity. However, ADP-β-S, an ADP analogue in which a terminal phosphate oxygen is replaced by sulphur, is known to be equipotent with ADP as an inhibitor of PGE1-stimulated adenylate cyclase in purified human platelet membranes. We therefore tested ADP-β-S on intact human platelets. ADP-β-S induced human platelet aggregation and inhibited PGE1-stimulated adenylate cyclase, but in botn cases was less potent than ADP and only achieved 75% and 50% respectively of the maximal effects of ADP. Aggregation induced by ADP-β-S was competitively inhibited by ATP (50 μM), a known ADP antagonist.

Both these actions of ADP could be inhibited by the simultaneous addition of ADP-β-S (50 μM). Aggregation induced by a stable endoperoxide analogue (11 ,9 -epoxymethano PGH2), which acts at a prostaglandin receptor rather than at an ADP receptor, was not inhibited by the simultaneous addition of ADP-β-S (50 μM). The behaviour of ADP-β-S towards human platelets is therefore tnat of a partial agonist at the ADP receptor.
Inhibition And Recovery Of Prostacycun And Thromboxane A After Low-Dose Aspirin
Authors: Preston F E , Jackson C A , Greaves M , Stoddard C J
Published: July 2018

We have compared the inhibitory effects of low-dose aspirin (ASA) on prostaglandin synthesis by vessel walls and platelets. Two groups of volunteers have been studied. Vein biopsies were obtained from five subjects before and at various intervals after 300 or 150 mg ASA. A further group of volunteers received aspirin 40 mg daily. In this group the second vein biopsy was taken three days after commencement of the drug. Prostacyclin synthesis was assessed by a radioimmunoassay (RIA) for 6-keto PGF1α. Platelet thromboxane A2 production was examined by RIA for thromboxane B2 (TXB2) 81-100 per cent inhibition of PgI2 synthesis was apparent 2 hours after 300 or 150 mg ASA. 86 per cent inhibition was still evident at 8 hrs. Simultaneously complete inhibition of platelet TXA2 production persisted more than 24 hrs. At this dose of ASA there is no difference in the initial inhibitory effect of aspirin on platelet and vessel wall cyclooxygenase. Also, in males the prolongation of the bleeding times following ASA does not appear to be the consequence of selective inhibition of TXA2. ASA 40 mg daily produced a cumulative inhibitory effect on TXA2 synthesis. Its effect PgI2 synthesis will be compared with that obtained after a single 300 mg dose ASA.
Some Biochemical Studies With SQ 26271: A Stable Partial Prostacyclin Agonist In The Human Platelet
Authors: Harris Don N , Phillips Marie B , Michel Inge M , Goldenberg Harold J , Heikes James E , Sprague Peter W , Antonaccio Michael J
Published: July 2018

A newly synthesized 9α-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26271 (8(S)9(S)11(R)12(S)-9,11-epoxy-9α- homo-5(Z),13(E)-15(R)-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation at concentrations which did not affect thromboxane B2 (TXB2) levels with an I50 value of 0.25 μM. It also was a potent inhibitor of platelet aggregation caused by collagen, 9,11- AzoPGH2, SQ 24810 (9,11-epoxy-9α-homo-5(Z),13(E)-15α- hydroxyprostadienoic acid) and epinephrine (secondary phase). However, it had little or no effect on thromboxane or prostaglandin synthetase activities. Moreover, SQ 26271 also inhibited the primary phase of epinephrine- and ADP- induced aggregation. In addition SQ 26271 caused a 3-fold increase in platelet adenylate cyclase activity, partially blocked prostacyclin stimulated adenylate cyclase activity and caused a 6-fold elevation of cyclic AMP levels in intact platelets, which was blocked by SQ 22536 (9-tetrahy- dro-2-furyl)adenine), an inhibitor of platelet adenylate cyclase activity. Finally, the inhibition of platelet aggregation by SQ 26271 was potentiated by an inhibitor of cyclic AMP phosphodiesterase (SQ 20009, etazolate), and reversed by SQ 22536. The above data indicate that SQ 26271 is a stable partial agonist of the prostacyclin receptor in human blood platelets.
Clinical Studies On Platelet Aggregation In Patients With Carcinoma Of Gastrointestinal Tract
Authors: Yamamura T , Matsumoto H , Maruyama Y , Wada T , Yamanaka M
Published: July 2018

Platelet aggregation was studied in 73 patients with carcinoma of gastrointestinal tract. Control group was composed of 45 patients with non malignant diseases. Aggregation was induced by adding 10μg/ml of Collagen, 1μ or 2μM at final concentration of ADP to that platelet rich plasma. Using aggregometer of Bryston, aggregation curves were recorded. The estimation of platelet aggregation was determined by aggregation rate, disaggregation rate and maximum aggregation rate. Patients with carcinoma were divided into two groups according to the presence of peritoneal dissemination, liver metastasis, lymphnode involvement as well as the depth of cancer invasion ( limited within submucosa layer or invaded below proper muscle ) and the size ( ≤ 5 cm or > 5 cm ). Moreover after resection of tumor, alteration in parameters of platelet aggregation was evaluated. Maximum aggregation rate was higher and disaggregation rate was lower in patients with carcinoma than in control group. There was significant differences in the intensity of platelet aggregation according to the rate of invasion and the size of the tumor, although peritoneal dissemination, liver metastasis, or lymphnode involvement did not affect platelet aggregation. Maximum aggregation rate and aggregation rate were higher and disaggregation rate was lower in the group with deeper invasion of tumor as well as in the group with larger tumors. After removal of tumor, platelet aggregation decreased. These results suggested that a substance or substances which activate platelet aggregation would be released from tumor tissue.
Isofocusing Used To Study The Specificity Of The “Lupus Inhibitor”
Authors: Coots M C , Glueck H I , Miller M A
Published: July 2018

This work was undertaken to investigate the clotting factor inhibition of the “Lupus Inhibitor”. Thiagarajan and Shapiro (Circulation 62: 279, 1980) showed that the “lupus anticoagulant" had immunologic specificity for the negatively charged phospholipids. However, in various case reports specific clotting factor inhibition has been described.

In our studies, the adsorbed, heat defibrinat- ed (56°C, 30 min.) plasmas of 6 patients with the “Lupus Inhibitor” were separated by preparative isofocusing. The inhibitory peaks were identified, and the clotting factor specificity of each was determined using partially purified eluates and specific factor deficient plasmas. The inhibitory peaks had activity against factors X, XI and XII in 5 of the 6 patients. In 1 patient, activity was confined to factor XI. These findings verified the initial factor quantitations. Utilizing the sequential Russell’s viper venom time, X-Xa was inhibited to a greater degree’ than the factor V and the phospholipid in the formation of prothrombinase. The celite eluate inhibition test (for XI, XII inhibition) was abnormal in all six patients.

Based on this study, the lupus inhibitor in these six patients was a heterogeneous collection of inhibitors directed against factors XII, XI and X rather than a homogeneous entity.
An Acquired Heparin-Like Anticoagulant In An 8 Month Old With Monoblastic Leukemia
Authors: Bussel J B , Steinherz P G , Miller D R
Published: July 2018

Well documented heparin-like anticoagulants have been reported in association with malignancy only in 3 adults with multiple myeloma. We report an 8 month old white male with acute monoblastic leukemia with an initial WBC of 358, 000/ul who was treated with adriamycin and ARA-C, developed a severe coagulopathy, and died 3 days later of presumed pulmonary and/or CNS hemorrhage. Liver and renal functions were normal and lysozyme was 112 mg/dl. The coagulation data were as follows:The factor V level was 11% and 2%, the factor VIII level was 69% and 85%, and the platelet count was approximately 50,000/μl both days. The thrombin time with mixing was 59 secs and after adsorption with “hepasorb” the thrombin time was 33 secs. Coagulation parameters did not change despite the infusion of fresh frozen plasma, proplex, hemofil, and platelets.

Additional studies revealed:
1) Progressive shortening of the thrombin time when increasing amounts of protamine and PF4 were added to patient’s plasma.
2) Patient’s urine prolonged the thrombin time of normal plasma. This effect was neutralized by protamine and PF4. Urine from another patient with AMOL in relapse did not prolong the thrombin time of normal plasma.
3) Mucopolysaccharides were detected in the patient’s urine by the Berry spot test.

The data suggest that the major coagulation abnormality in this patient was a circulating heparin-like anticoagulant. This is the first case report of an endogenous heparin-like anticoagulant in leukemia.
Procoagulant (Tissue Factor) Activity Is Higher In Cells From Murine Sarcoma Sublines With Lower Metastatic Potential
Authors: Colucci M , Giavazzi R , Alessandri G , Semeraro N , Mantovani A , Donati M B
Published: July 2018

It has been suggested that cancer cell procoagulant activity influences metastasis formation by promoting fibrin deposition around tumors. We have investigated the procoagulant activity of various tumor cell sublines with different metastatic capacity. These were derived from spontaneous lung nodules of mFS6, a benzopyrene-induced sarcoma in C57B1/6 mice. After one in vivo passage by s.c. implantation, the resulting tumor was cultured once in vitro till confluence; cells were then harvested from plastic bottles by trypsin treatment, and washed extensively after trypsin neutralization. Tumor cell procoagulant activity was measured by a one-stage clotting assay using autologous plasma. All the cells tested possessed thromboplastin-like activity since they shortened the recalcification time of normal and factor VUI-deficient plasma to a similar extent but had no activity on factor Vll-deficient plasma.

They were, however, heterogeneous as regards the degree of procoagulant activity; the two cell lines with virtually no metastatic capacity showed 6-8 times higher procoagulant activity than the cells from the parent line; in contrast, the procoagulant activity of the two sublines with higher metastatic capacity did not differ significantly from that of the parent line.

These findings support the hypothesis that fibrin is part of a defence reaction against cancer cell invasiveness.
Cancer Procoagulant: A Novel Vitamin K-Dependent Activity?
Authors: Donati M B , Delaini F , Colucci M , Vitti G De Beilis , Locati D , Poggi A , Semeraro N
Published: July 2018

Cells from some experimental tumors have been found to possess a peculiar procoagulant activity, which directly activates coagulation factor X, independently of both the intrinsic and extrinsic clotting pathways. We report here that, in the Lewis Lung Carcinoma (3LL) model, such a cell procoagulant is inhibited by vitamin K-deficiency (either dietary or pharmacologically induced).

Warfarin was given chronically to mice in drinking water from day 7 after i.m. tumor implantation at a schedule which maintained the prothrombin complex activity (thrombotest) at less than 5% of control values. Vitamin K-deficiency was also induced by feeding the mice with an appropriate diet at a schedule which gave a similar degree of plasma anticoagulation. At day 18 after tumor implantation macrophage- free 3LL cell suspensions obtained by spilling of necrosis- free tumor fragments were tested for their specific clot- promoting capacity by a one-stage recalcification time of factor VII-deficient plasma. 3LL cells from either group of vitamin K-deficient animals gave significantly longer clotting times than controls (84 ± 4 and 82 ± 3 sec versus 42 ± 2 sec, p< 0.001, n = 15 for each of the 3 groups) corresponding to 18-25% of the activity of controls.

This effect was completely reversed by adding vit. K to drinking water of vitamin K-deficient animals. Administration of autologous prothrombin complex concentrate, sufficient to completely correct the thrombotest values for several hours before tumor cell harvest, did not modify the effect induced by vitamin K-deficiency on 3LL cells. This data suggests that cancer procoagulant may represent a new vitamin K-dependent activity; vitamin K-deficiency would thus display not only a plasmatic but also a “cellular” anticoagulant effect. The latter could be relevant to the reported antimetastatic effect of vitamin K-deficiency.
Distribution And Localization Of Urinary Procoagulant In The Human Kidney And Its Immunologically Cross-Reactive Substances In Various Exocrine Glands
Authors: Matsuda M , Aoki N
Published: July 2018

Procoagulant purified from human urine (urinary procoagulant, UP) was found to be macroaggregates with a molecular weight of more than 2.0 × 107 on the basis of gel exclusion chromatography on Sepharose 4b. UP does not function as a proteolytic enzyme, but does facilitate the factor VIIa-catalyzed activation of factor X in the presence of calcium ions, thus it may be categorized as tissue factor. Similar procoagulant activity was found also in various secreta including milk, saliva, bile, gastric juice, pancreatic juice and sweat, in this order of potency. The activity in milk and saliva, for example, was 14 and 5 times as potent as urine, respectively. Monospecific antiserum against UP and its IgG fraction inhibited efficiently the procoagulant activity of these secreta as well as UP.

An immunohistochemical study using the anti-UP antiserum revealed that the immunologically cross-reactive substances were distributed in the epithelial cells of these exocrine glands. In the kidney UP was distributed to the loop of Henle and distal convoluted tubules. In these areas UP was found in association with the luminal and intercellular borders as well as in the cytoplasm of epithelial cells. Both the descending and ascending limbs of Henle were equally stained. The cytoplasmic staining was patchy in distribution among cells of distal tubules and was predominantly localized in the supranuclear areas. Casts in the distal tubules were also positively stained.

These findings suggest that UP is synthesized in the epithelial cells of these particular parts of nephron and is secreted into urine. It is also suggested that the immunologically cross-reactive substances are synthesized in the epithelial cells in various exocrine glands and are excreted into secreta.
Purification Of Rabbit Leukocyte Tissue Factor (Thromboplastin (LTF)
Authors: Kang Sungzong , Niemetz Julian
Published: July 2018

A variety of stimuli, including bacterial endotoxin, may endow leukocytes with potent procoagulent (tissue factor) activity (TFa). The characterization and purification of this activity was undertaken. Microsomal membrane fractions of endotoxin-stimulated rabbit macrophages were obtained by standard procedure of disruptions and stepwise centrifugations. The membrane fragments containing the highest tissue factor activity were solubilized in sodium barbiturate- deoxycholate buffer (0.05M-0.5%) at pH 8.0 and centrifuged at 180,000 × g for one hour. The concentrated supernatants were subjected to Sephacryl-200 chromatography and eluted with a barbiturate-deoxycholate buffer (0.05M-0.5%). The chromatography dissociated phospholipids from tissue factor apoprotein with apparent loss of activity ensuing. The fractions with high tissue factor activity were pooled and concentrated. Sodium dodecyl sulfate (SDS) was added to the concentrated fraction to a final concentration of 2%. The mixtures were further separated by preparative polyacrylamide gel electrophoresis (prep PAGE). Rabbit brain tissue factor was prepared similarly. Contrasting with rabbit brain preparations, where the TFa eluted as a single peak from prep PAGE, the leukocyte TFa eluted from prep PAGE as 3 or 4 peaks. On analytical PAGE, the different peaks showed slightly different mobilities, the tissue factor activity migrating discretely with each band. A molecular weight of 43,000 was derived for both the fastest band of leukocyte TF and brain TF. These findings suggest different molecular subspecies for rabbit leukocyte TF. (Supported in part by grants from NIH and the Veterans Administration.)
Involvement Of Human Factor IX In Tissue Thromboplastin Induced Coagulation
Authors: van den Besselaar A M H P , Ram I E , Bertina R M
Published: July 2018

This study is concerned with the question whether activation of factor IX by factor VII - tissue thromboplastin contributes to the rate of plasma coagulation. The protein component of tissue factor was partially purified from human brain. Its molecular weight as deduced from SDS - polyacrylamide gel electrophoresis was about 48,000. Reconstitution of thromboplastin activity was obtained by mixing apoprotein and phospholipids in the presence of Triton X-100 and subsequent removal of Triton by adsorption to Biobeads SM-2. Reconstituted tissue factor greatly accelerated the activation of factor IX by isolated factor VII in the presence of calcium ions. In a contact free system (plasma from a patient with congenital factor XII deficiency; factor XII<0.001 Unit/ml) plasma coagulation times (tc) were determined as a function of apoprotein concentration (at constant phospholipid) both in the presence and absence of factor IX. At high apoprotein concentration tc showed to be independent of factor IX, whereas at low apoprotein concentration the removal of factor IX resulted in a 2 - 3 fold increase of tc. The involvement of the tissue factor - factor VII complex in this phenomenon was evaluated using a specific anti-factor VII serum. The results indicate that activation of factor IX by factor VII - tissue thromboplastin does not significantly contribute to the rate of plasma coagulation.