bluuebunny/crossref_metadata_2025 huggingface dataset:
Incorporation Of3H-Glycerol into Platelet Phospholipids During Platelet Aggregation
Authors: Hutton R.A. , Hardisty R.M.
Published: May 2019

In vitro incorporation of glycerol-2-3H into phospholipids of normal platelets was measured at rest and during platelet aggregation induced by adenosine diphosphate, epinephrine, collagen and ristocetin. Compared to the basal state, aggregating platelets showed a marked increase in total glycerol uptake, although total platelet phospholipid content was unchanged. The most striking change was an increase in the proportion of the radioactivity incorporated into the phosphatidyl inositol fraction (from 11% to 36% after 30 minutes incubation with epinephrine), this being largely at the expense of the phosphatidyl choline fraction which decreased from 46% to 30%. The extent of the glycerol uptake correlated well with the degree of platelet aggregation observed (p = 0.05 for adenosine diphosphate), but was not directly related to the speed of either aggregation or the release reaction. The rate of glycerol uptake paralleled the development of platelet factor 3 availability (PF3a) over the first 20-30 minutes of incubation, but thereafter, PF3a levelled off while glycerol uptake continued to rise for at least another 30 minutes. We conclude that the changes in platelet phospholipid turnover observed here during platelet aggregation, are of little direct consequence to the cells haemostatic functions. The increase in phosphatidyl inositol turnover may represent part of the cells’ response to membrane distortion or damage during the secretory process, as has been documented in other secretory processes.
Stimulation of Monocyte Procoagulant Activity by Adherence to Different Surfaces
Authors: Ginkel C.J.W. van , Oh J.I.H. , Aken W.G. van , Vreeken J.
Published: May 2019

When mononuclear leukocyte suspensions (5.109 cells/1, 25% monocytes) were incubated on glass dishes, 57 ± 1% of the monocytes were found to adhere to the surface after 3.5 h incubation at 37°C. It was found that the adherent cells shortened the recalcification time from 435 ± 35 to 50 + 2 sec using normal plasma as substrate. However, when monocyte adherence was prevented by incubating the leukocytes on cuprophane (3 + 1% adherence), much less PCA was detectable (the recalcification time was shortened from 435 ± 35 to 201 ± 19 sec). After exposing to glass the non-adherent monocytes had negligible PCA in comparison to the adherent monocytes. Protein synthesis inhibitors (cycloheximide and actinomycin D) inhibited PCA generation but did not affect monocyte adherence. These data provide the first evidence that adherence of monocytes is a stimulus for the generation of thromboplastic activity.
An Enzyme in Rabbit Aorta that May Play a Role in Haemostasis and Thrombosis
Authors: Lieberman G.E. , Lewis G.P.
Published: May 2019

Platelet interaction with blood vessel walls has been studied with the intention of elucidating regulatory mechanisms involved in haemostasis and thrombosis. Microsomal fractions (Mif) prepared from rabbit aortas have been found to inhibit ADP-induced platelet aggregation when incubated with ADP prior to addition to platelet-rich plasma. Thin layer chromatography of incubation mixtures in which [8-14C] ADP has been used has proved that the inhibition is due to degradation of ADP. The main degradation product at pH 6.0 is AMP and between the pH optimum of 7.2-8.2, it is adenosine. The rate of ADP breakdown is dependent on the duration, temperature and pH of the incubation, and on the concentrations of ADP and Mif used. Enzymic activity can be destroyed by boiling and by treatment with perchloric acid.EDTA (Na2) will remove inhibitory activity but this can be restored by addition of either calcium or magnesium to the incubate. The subcellular localization of the enzyme has been determined by isopycnic centrlfugation in a sucrose density gradient, using marker enzymes to establish the distribution of subcellular organelles in the gradient.ADP degrading activity was found to coincide with the peak of 5′ nucleotidase (5′NT) activity, the plasma membrane marker.

Localization of the ADP degrading enzyme on the cell membrane means that it is in a position to destroy any ADP which might be released by platelets in their response to damage of the blood vessel wall. Its coincidental location with means that any AMP formed will be converted to adenosine which is itself an inhibitor of platelet aggregation. The ADP degrading enzyme could therefore play an important role in limiting the extent of thrombus formation.
Antithrombin Functional Activity in Normal Individuals and in Patients with Familial Hypercholesterolemia and Nephrotic Syndrome
Authors: Zucker M. , Gomperts E.D. , Russel D. , Salant D. , Feesey M. , Seftel H. , Mendelsohn D.
Published: May 2019

Nineteen patients with familial hypercholesterolemia, 5 patients with hyperlipidaemia secondary to nephrotic syndrome and 14 normal individuals were given a mean containing lg saturated fat/Kg body weight. The plasma total functional antithrombin (AT) activity was measured in the normal before the meal and 2,4 and 6 hours after the meal by both a clotting assay system and the chromogenic substrate S2160. Only the clotting assay system could be employed in the hyperlipidaemia situation. The functional plasma AT-III activity was compared with AT protein levels measured by quantitative radial immunodiffusion. Functional AT activity was depressed maximally at 2 hours after the meal in 13 of the normal and all of the nephrotic individuals studied. The immunologically measured AT-III levels remained constant throughout the experiment. Only 9 of the 19 familial hypercholesterolemic patients showed a similar phenomenon. The functional depression in AT activity failed to correlate with the rise in serum triglyceride level. Basal functional AT activity was similar in the normal and hypercholesterolemia patients but considerably increased in the nephrotic patients studied. When similar studies with an unsaturated fatty meal were carried out in normal volunteers no significant alteration in functional AT activity was observed. In addition, 11 volunteer normal individuals were fasted for 24 hours. Considerable increases in functional AT activity were present in 9 of the 11 subjects. These studies indicate that functional AT activity may be modified by various physiological and pathological factors.
Large Scale Method for the Isolation of Antithrombin III.
Authors: Wickerhauser M. , Williams C.
Published: May 2019

Antithrombin III (AT III) concentrates may be of value in the treatment of various hypercoagulable states associated with congenital or acquired AT III deficiency. We have developed a large scale method for isolation of AT III from plasma or from Cohn fraction IV-1, an unused byproduct of routineplasma fractionation. The method consists of the following steps:(a) precipitation of the starting plasma or Cohn fraction IV-1 extract with 2 0% polyethylene glycol (PEG)4000 to remove a number of impurities including hepatitis B antigen if present;(b) batchwise adsorption to and elution from heparin-Sepharose using one volume of gel for each 50 liters of the 20% PEG supernatant; and (c) concentration of the eluted AT III by bulk lyophilization, followed by desalting on Sephadex G-50, sterile filtration and final lyophilization. In two large scale experiments, 7.1 and 8.7 grams of AT III, determined as antigen by radial immunodiffusion, corresponding to 25,000 and 31,000 plasma equivalents, were recovered from 100 liter plasma and 15 kg Cohn fraction IV-1 batches respectively. Both preparations were over 95% pure by disc gel electrophoresis and had an activity to antigen ratio close to that of AT III in plasma. Both preparations were nonpyrogenic and met all other FDA requirements for biologic products.
β-Thromboglobulin and Deep vein Thrombosis in Surgical Patients
Authors: Smith RC , Ruckley CV , Duncanson Jeanette , Allan NC , Dawes Joan , Pepper DS , Cash JD
Published: May 2019

Preliminary studies suggested that β-Thromboglobulin (βTG)assay might be of diagnostic value in venous thrombosis(DVT). This study examinedTG levels in 16 normal subjects, 2 4 patients presenting with phlebographically proven DVT or pulmonary embolism (PE)confirmed by lung scan and 46 patients screened byI-fibrinogen test (IFT)for post-operative DVT. The mean in 46 normal samples was 33 ng/ml ± SD 19 ng/ml, only two had levels above 70 ng/mls. 12 of 24 patients presenting with DVT/PE had values >70 ng/ml on presentation and all but three had values of >70 at some stage during subsequent daily sampling. The onset of DVT as detected by IFT in 13 patients was associated with a mean rise in βTG but the difference was not significantly different from normal values. We confirm the association between elevated βTG and venous thrombosis but the variability of the results with current sampling techniques limit its value as a diagnostic test.
The Organization of Dutch Thrombosis Services
Authors: van Dijk-Wierda C.A. , van Dijk J.L.
Published: May 2019

In the Netherlands the prevention and treatment of thrombotic diseases by means of coumarin derivatives are carried out by “Thrombosis Services”, which cover a territory of 9 million inhabitants where they perform more than 2 million prothrombin estimations a year. A thrombosis service is an institute with the object to an appropriate treatment of patients with oral anticoagulants by means of:providing dosage and other advices by expert doctors attached to the service;venapuncture by skilled nurses at the patients home or at out-patient departments and standardized laboratory work. The Thrombotest of Owren is used for thelaboratory assay, aiming at therapeuticlevels of 5-10%. In a well organized thrombosisservice a score of 83% of thelaboratory results within the target limits are made. In 1971the thrombosis services were united in a federation whichset itself to guality control and improvement by means of standardization of techniques in laboratorycontrol, directivesfor dosage, instruction to patients, external quality control, etc. This highly privileged condition seems one of the reasons that the Netherlands do notsharethe negative attitude towards the efficiency of coumarin described in many other countries.
Fibrinopeptide a Measurements in the Study of Thrombosis
Authors: Nossel H.L.
Published: May 2019

Elevated plasma fibrinopeptide A (FPA) levels have been found in all patients with acute (symptoms for 5 days or less) venous or arterial thrombosis and/or embolism studied in our institution. Heparin therapy results in a decline of fibrinopeptide A levels to the normal range. In most instances the normal level is attained within 15 minutes of the start of therapy but in some instances return to normal is delayed for up to 72 hours.FPA levels have not been tested in a formal study on the prediction of thrombosis. The results will be presented of serial FPA measurements and the relationship of FPA elevations to recurrence of symptoms and. extension of the disease in patients with thrombo-embolism. The usefulness of FPA tests in the diagnosis of prethrombotic states will depend on the pathophysiology of these states.

Measurement of FPA levels may be useful in predicting thrombosis under circumstances in which activation of the coagulation system precedes thrombosis. When thrombosis results from the reaction of normal blood with diseased vessel wall FPA levels will not be useful in prediction.Since- the pathogenesis of thrombosis is likely to vary in different syndromes, the pathophysiology of these syndromes will have to be investigated separately to answer questions as to the usefulness of such tests in prediction.
Thrombin-Antithrombin III and Plasm in-Antiplasmin Complexes as Indicators of in Vivo Activation of the Coagulation and/or Fibrinolytic Systems
Authors: Collen D. , Cock F.de
Published: May 2019

Both the thrombin-antithrombin III and plasmin-antiplasmin complexes contain neoantigenic structures, not present in the parent molecules, to which non cross-reacting precipitating antibodies can be produced (Thromb.Res. 5, 577, 1974). This phenomenon has been used to develop immunochemical methods for the quantitation of these complexes in human blood (Thromb. Res 7, 235, 1975). Patients with in vivo activation of the coagulation and/or fibrinolytic systems consistently showed increased levels of enzyme-inhibitor complexes (Eur.J.Clin.Invest, in press, 1977). The circulatory half-life of these complexes was estimated to be several hours (0.5 to 0.75 days).

On several occasions, increased plasmin-antiplasmin titers were observed in the presence of a normal screening hemostasis analysis, suggesting that these assays may be more sensitive indicators of in vivo activation of the coagulation-fibrinolytic systems that assays for fibrinogen degradation products.

Studies to determine the clinical value of the measurement of these enzyme-inhibitor complexes as indicators of a prethrombotic state are in progress and will be reported.
Spontaneous Platelet Aggregation in Vivo
Authors: Hoak J.C.
Published: May 2019

In recent years several new methods have been developed to quantitate the presence of platelet aggregates in whole blood. Included are techniques using an electronic particle analyzer after lysis of the RBC’s with saponin, fixation of platelet aggregates with dilute formalin, and measurement of changes in filtration pressure in systems where platelet aggregates obstruct the flow of blood across standardized filters.

Using the formalin-fixation method (Lancet 2:294, 1974) to detect platelet aggregates we have found abnormal values in patients with transient ischemic attacks, acute myocardial infarction, acute peripheral arterial insufficiency, some patients with recurrent deep vein thrombosis, migraine, and certain types of ischemic retinopathy. Questions remain, however, as to whether the detected aggregates are definitely circulating aggregates or whether the technique selects out a population of abnormal platelets which behave differently upon exposure to formalin than to the normal platelets. While the current method appears to be satisfactory to delineate patients in whom platelet aggregates may be playing a primary or secondary role, additional information is required to provide a satisfactory explanation of the mechanisms involved if we are to achieve an optimal application of the technique.
The Clinical Interpretation of Platelet Survival with Cr51 Tagged Platelets
Authors: Jonker J.J.C. , van Riel L.H.M. , Schopman W. , den Ottolander G.J.H.
Published: May 2019

We obtained experience in 800 determinations of platelet survival time(PST) with a modified method according to Aster and Jandl . In 20 normal volunteers we obtained a mean T½ of 99 hours with a S.D. of 13.4 hours. The reproducibility of the method was obtained from two PST measurements with an interval of one week. The S.D. of replication was 13.4 percent. The reproducibility of PST over a long period of time (18-30 months) was determined from patients of the placebo group of a trialon patients with angina pectoris. Compared with the PST at entry in 72 patients the mean standard deviation in at least three determinations was 14.5%. Each single PST has a range of 90% reliability (T½ ± 2x S.D.) depending on the correlation factor between disappearance in comparison with time. Our mean correlation factor (R) is 0.92 with a range of 0.85-1.00). A single PST with a R of 0.92 has an S.D. of 15%, meaning that there is a significant difference between two PST determinations in a single individual when there is more than 30% difference. At entry 30% of angina patients has a shortened PST. After six months there was an increase of the PST in the Clofibrate treated group. After 18 months however there was also an increase in the placebo group. This means that no trial of drug effects on platelet survival can be accepted which are lacking a double blind setting and a control group.
Factor X Activation in Sodium Citrate
Authors: Aronson D.L.
Published: May 2019

Early studies showed that partially purified prothrombin preparations generated thrombin in solutions of (NH4)2SO4 or sodium citrate. Milstone established that-the prothrombin activating enzyme Thrombokinase (Xa) would activate in (NH4)2SO4. More recent studies of the physiologic activation of Factor X have defined a role for both the intrinsic and extrinsic coagulation pathways. It has been proposed that the citrate activation of Factor X is independent of Factor VII or IX and results from the direct action of either thrombin or Xa on Factor X.

When Factor X is incubated in sodium citrate there is a lag phase followed by the rapid generation of Factor Xa. This is followed by the degradation of Xa to a form which cannot interact with the cofactors of prothrombin conversion. If DFP is added during the phase of rapid X generation the reaction is inhibited. The addition of DFP prior to the activation has no effect.SBTI totally inhibits the activation of Factor X. Removal of residual Factor VII from the Factor X preparations prevents citrate activation and readdition of Factor VII restores this capability. Preincubation of the Factor VII in citrate does not effect the lag phase and the addition of Factor Xa to VII deficient Factor X has minimal effect.

It is proposed that citrate activation of Factor X is Factor VII dependent but that the Factor VII must be activated by Factor Xa (or possibly thrombin). The inability of the degraded form of Xa to react with the cofactors may be due to the loss of the γ-carboxy glutamate containing portions of the molecule.
Prototypes for Clotting by Transamidation
Authors: Myhrman R.V. , Lorand L.
Published: May 2019

In the plasma of vertebrates, onset of clotting reflects on the ability of thrombin to hydrolyze fibrinogen; stiffening of the clot network by transamidation is a later event. We will contrast this sequence with two entirely different systems in which clotting occurs apparently by transamidation alone. The formation of the copulation plug from guinea pig seminal vesicle secretion (I) and the coagulation of lobster blood plasma (II) will be considered. Whereas in vertebrate blood the zymogen (Fibrin stabilizing factor or Factor XIII) co-circulates with fibrinogen, the other two enzymes are sequestered from their clotting substrates by being stored in separate tissues (coagulation gland in case I, amebocyte or muscle in case II). Clotting of guinea pig seminal vesicle secretion (Williams-Ashman et al., Proc. Natl. Acad. Sei., 69, 2322, 1972); purification and properties of a transamidase from the guinea pig prostate (Wing et al., Fed. Proc., 33, 290, 1974); release of the lobster amebocyte enzyme; its purification from lobster muscle together with its enzymatic properties; and the use of the lobster clotting system in the diagnosis of a human defect of fibrin stabilization (Lorand et al., J. Clin. Inv., 47, 268, 1968) will be reviewed.
Ca2+-Induced Cleavage of Human Platelet Polypeptides Mediated by the Calcium Ionophore A-23187
Authors: Jakábová Milica , Phillips David R.
Published: May 2019

The effect of calcium on human platelet polypeptides was investigated. When lysed platelets were incubated with mM Ca++, two major intracellular polypeptides (Mr = 255,000 and 230,000) were found to rapidly disappear. A similar phenomenon was also observed when intact platelets were treated with the calcium ionophore A-23187 in the presence of mM Ca++. Determinations of lactic dehydrogenase activity in supernatant fractions demonstrated that these losses occurred before platelet lysis. Investigations into the identity of the high molecular weight polypeptides revealed that one (Mr = 255,000) had similar properties to actin binding protein. The loss of the high molecular weight polypeptides was accompanied by formation of lower molecular weight polypeptides (Mr = 135,000, 93,000 and 48,000), indicating that Ca++ activates a polypeptide cleavage mechanism. The Ca++-activated polypeptide cleavages were rapid, with significant changes being observed within the first 0.5 min of incubation. An obvious explanation for these effects is. that there is Ca++-activated proteolytic activity within platelets. The Ca++-activated proteolytic activity was determined by the hydrolysis of the artificial substrate azocasein. We found that more than 90% of the proteolytic activity in lysed platelets was due to Ca++-activated proteases. These studies show that Ca++-activated proteases may play an important role in platelet activation.
High Gene Frequency of Factor XI Deficiency in Ashkenazi Jews
Authors: Seligsohn Uri
Published: May 2019

Factor XI (fXI) deficiency has previously been reported mainly in Jews. Its frequency and ethnic distribution however, have not been determined. It was the purpose of this study to examine these questions.

Since 1966, 78 patients with fXI deficiency have been observed. They belong to 36 unrelated families, all of whom are of Eastern European origin (Ashkenazim).

Of 428 normal Ashkenazi Jewish subjects whose fXI levels were assayed, 1 subject had severe deficiency (3%), 35 partial deficiency (24 - 49%) and 392 had normal levels. The 95% confidence limits of the frequency of severe fXI deficient subjects in the total Ashkenazi Jewish population are 0.1 - 0.3%, and for partial deficient subjects 5.5 - 11%.

Since severe as well as partial fXI deficient patients may not be diagnosed until profuse bleeding is presented following trauma, the observed high gene frequency warrants performing the appropriate tests in all Ashkenazi Jewish patients undergoing surgery.
The Generation of Bradykinin in Clinical Blood Samples
Authors: van Rosevelt R.F. , Philipse-Hoorweg E.M. , Mulder A. , van Mourik J.A.
Published: May 2019

Kallikrein specifically cleaves bradykinin (BK) from high molecular weight kininogen. Although measurement of BK in blood samples is potentially a sensitive index of intravascularkallilirein action, the rapid in activation of BK in the circulation(t

=20 sec) limits the practical usefulness of BK assays. The present study was designed to develop a method which permits rapid measurement of circulating kallikrein in a large number of samples. Blood is collected in a mixture of kininase inhibitors and incubated for different periods of time before addition of soybean- and ovomucoid trypsin inhibiter. BK was measured by a radioimmunoassay. Cross-reacting kininogens were quantitatively separated from BK by precipitation with polyethyleneglycol. In 25 normal individuals this assay revealed a mean BK plasma level of 0.5 ng/ml±0.5(2SD) and the BK generation In vitro (ΔBK) of 1.5 ng/ml/20 min ± 1.0 (2SD). Upon activation of plasma with kaolin or dextransulphate, BK increased up to 800 ng/ml/20 min. In 3 out of 5 patients with asymptomatic hereditary Cl-INH deficiency, ΔBK was increased (range 7.0-11.1 ng/ml/20 min) whereas basal BK levels were normal. Increased ΔBK was found in 3 patients with pancreatitis and in 9 out of 26 patients with a malignancy (range 2.8+13.8 ng/ml/20 min)the basal BK plasma levels being normal. 2 of the latter patients showed signs of low grade DIC. It is thought that the BK-generation test detects circulating kallikrein.
Factor XII Deficiency and Thrombosis
Authors: Pizzuto J. , García N. , Reyna MP. , Conte G. , Ambríz R.
Published: May 2019

The clinical implication of Factor XII deficiency has been much discussed. Most authors agree that these patients are generally asymptomatic; others have found a bleeding tendency; and some 15 scattered cases with occlusive vascular disease have heen collected from world’ literature. Since 1972 we have studied 8 patients with this deficiency associated with arterial or venous thromboses repeated in 4 of the 8, in different sites; all male, median age 33.6 (range 18 to 65) with no known risk factors for the development of thrombosis. The average activity of factor XII in the 8 patients was 38 %, comparable with that reported in earlier studies (Thrombos Diathes Haemorrh 23: 423, 1970), and that of 20 normal subjects of similar ages with 182 % (p < 0.0001). In conclusion, Factor XII deficiency may in some cases be considered a risk factor for thrombosis. or, at least a lack of protection against its development.
Morphological Aspects of Haemostasis in the Uterus During Menstruation
Authors: Sixma J.J. , Christiaens G.C.M.L. , Haspels A.A.
Published: May 2019

Light and electron microscopical studies were performed on 8 normal uteri extirpated during the first 24 hours of menstruation. In the premenstrual phase with clinical spotting stromal desintegration had already occurred. Vessel lesion sometimes with open communication between the vessel lumen and the surrounding stroma, but without hemostatic reaction were observed. During early menstruation (up to 7 hrs after the onset of menstruation) extravasation of blood was observed in the superficial layers of the functional is. Vessels in this zone were plugged by thrombi containing variable amounts of platelets and fibrin. These thrombi were desquamated with the shed tissue. At 13 hours after the onset of menstruation the functional layer was almost fully desquamated. Few thrombi with a relatively fresh appearance were detected at the transition of basal into functional layer. From 20-72 hrs after the onset (late menstruation) almost all functional endometrium was shed. No extravasation of blood and no intravascular thrombi were observed. Hemoscasis may be caused by adherence of vessel lips. This study is the first study to indicate the direct involvement of platelets and fibrin in hemostasis in the menstruating uterus. Ihe differences with hemostasis elsewhere in the body is very pronounced.
Gel Filtration of 125I-fibrin and 131I-fibrinogen at 20°C and at 37°C
Authors: Krell W. , Mahn I. , Müller-Berqhaus G.
Published: May 2019

Sepharose-6B columns were used to investigate the behaviour of urea-solu-bilized 125I-fibrin during gel filtration. Urea-solubilized 125I-fibrin was mixed with rabbit plasma containing 131I-fibrinogen. This mixture was gel-filtered at 20°C and 37°C. The elution medium was a TRIS-PO4 buffer, pH 7.4, containing EDTA, EACA and aprotinin. Results: 125I-fibrin was separated from 131I-fibrinogen at 20°C as well as at 37°C. Only 3% of the 131I-fibrinogen was eluted tcgether with fibrin at 20°C. At 37°C, however, no 131I-fibrinogen was eluted together with 125I-fibrin. At 370°C a mean of 90% of the applied 125I-fibrin was retained on the columns, whereas at 20°C a mean of 40% of the 125I-fibrin was retained. At both temperatures, 80-90% of the 131I-fibrinogen was eluted. From these experiments we con-clude:1. If 125I-fibrin is chromatographed on a TFIS-PO4 medium it partially precipitates on the column. 2. Fibrinogen and/or other plasma proteins are necessary to keep fibrin in solution. 3. Interactions between fibrinogen and fibrin are not maintained during gel filtration in a buffer medium, 4. Possibly formed complexes at 20°C do not exist at 37°C.

(Supported by the Deutsche Forschungsgemeinschaft).
The Effect of Antihuman Platelet Antibodies on Human Platelets
Authors: Ali-Briggs E.F. , van Leeuwen E.F. , von dem Borne A.E.G. , Jenkit C.S.P.
Published: May 2019

The influence of an heterologous antihuman platelet serum (AS) was examined an immunological approach to investigate platelet function, as induces aggregation and release from platelets in citrate plasma. Neither thrombin nor complement were requirements, and inhibitors of ADP-(adenosine) and collagen-induced (aspirin) aggregation were only slightly inhibitory. EDTA-P and four-times washed platelets were also aggregated by as. These data sugf AS-induced aggregation proceeds, at least in part, by a mechanism independer of adp and does not involve platelet prostaglandin synthesis. The active orinciple of the as was found to be associated with the IgG fraction. as fo ipmunoprecipitates (by immunodiffusion) with platelets, fibrinogen and facte VIII; absorbtion with cryoprecipitate did not influence the aggregation resf Since platelets have common antigenic determinants with other blood cells, absorbtion studies with such cells were performed. Absorbtion of as with cells (until negative in the indirect antiglobulin test) had little effect of AS–induced aggregation and release. Following absorbtion with leucocytes, immunofluorescence studies revealed that lymphocyte reactive antibodies were almost totally removed while those of the granulocyte and platelet were stil present. This absorbed as retained the ability to induce both platelet aggregation and release.
Evaluation of an Automated Amidolytic Antiplasmin Assay
Authors: Kahlé L.H. , Lamping R.J. , tenCate J.W.
Published: May 2019

The manual antiplasmin (AP) assay (Teger-Nilsson, Scand.J. Clin. Lab. Invest. 1977) has been adapted for automatically performance on an Automated Kinetic Enzyme and Substrate analyzer. Comparison of the automated method with the manual method revealed good correlation (r=0.98). No significant differences were found when EDTA or citrate plasmas were used. Normal values from 160 healthy individuals were 101% (79%-125%). Addition of purified AT III to plasma had no effect on the AP assay. Also no effect was observed after infusion of AT III concentrates in patients with decreased AT III levels. In patients with liver cirrhosis AP levels were 74% ± 14%, which were significantly higher than their AT III levels (62% ± 12%). The same was observed in newborns, where the mean AP activity was 81% ± 17%, while their AT III activities were 49% ± 15%. From the AP assay in 985 patients only 5% (52) showed a decreased level (below 80%) and only 3% below 70%. For the AT III activities these figures were 20% and 12% respectively. Assay of both AP and AT III activity discriminates between Dissiminated Intravascular Coagulation and systhemic fibrinolysis.
Fluorogenic Peptide Substrates for Proteases in Blood Coagulation, Kallikrein-Kinin and Fibrinolysis Systems: Substrate for Plasmin and Factor XIa
Authors: Iwanaga S. , Kato H. , Maruyama I. , Adachi N. , Ohno Y. , Takada K. , Kimura T. , Sakakibara S.
Published: May 2019

Fluorogenic peptide substrates, peptidyl-4-methyIcoumarin amides (MCA), are now commercially available and proved to be useful for the assay of α-thrombin, Factor Xa, urokinase, Limulus clotting enzyme, plasma and urinary kallikreins. We have newly synthesized the fluorogenic substrates for plasmin and Factor XIa and tested for their possible use as specific substrates for these enzymes. Among six peptidyl-Lys-MCA, Boc-Clu-Lys-Lys-MCA and Boc-Val-Leu-Lys-MCA were good substrates for bovine and human plasmins, which were activated by urokinase. Streptokinase-activated plasmin complex (molar ratio with 1 to 1) hydrolyzed the former but not the latter. Using Boc-Phe-Ser-Arg-MCA and Boc-Leu-Thr-Arg-MCA, a specific assay method for Factor XIa activated with Factor XIIain the presence of HMW kininogen and kaolin, was developed. The kinetic parameters of various proteinases towards the specific substrates were as follows: α-Thrombin (Boc-Val-Pro-Arg-MCA, Km=2.1×10-5M), Factor Xa (Boc-Ile-Glu-Gly-Arg-MCA, 1.6×10-4M), Plasma kallikrein (Z-Phe-Arg-MCA, 2.4×10-4M), Pancreatic kallikrein (Pro-Phe-Arg-MCA, 1.6×10-4M), Urinary kallikrein (Pro-Phe-Arg-MCA, 2.2x10-4M), Urokinase (Glutaryl -Gly-Arg-MCA, 4.4×10-4), Limulus clotting enzyme (Boc-Leu-Gly-Arg-MCA, 2.7×10-5M), Plasmin (Boc-Glu-Lys-Lys-MCA, 6.7×10-5M and Boc-Val-Leu-Lys-MCA, 2.5×10-4M).
Investigations of DES-AB I-125 Fibrin and I-131 Fibrinogen by Agarose-Plasma Chromatography at 20°C and 37°C
Authors: Mahn I. , Krell W. , Müller-Berqhaus G.
Published: May 2019

Human des-AB I-125 fibrin was prepared by clotting I-125 fibrinogen with thrombin in the presence of EDTA and aprotinin, solubilized in buffered 3 M urea and mixed with plasma containing I-131 fibrinogen. The molecular ratio of fibrin to fibrinogen was 1:20. These fibrin-fibrinogen mixtures in plasma were applied to sepharose CL-6B columns equilibrated and eluted with plasma at 20°C and at 37°c (Agarose-plasma columns). I-125 fibrin and I-131 fibrinogen could completely be recovered from the columns at both temperatures. I-125 fibrin was separated from I-131 fibrinogen at 20°C as well as at 37°C. I-125 fibrin was eluted in the void volume in front of the I-131 fibrinogen. At 20°C, a mean of 5.8 % of the I-131 fibrinogen at 37°C, however, only a mean of 1.2 % of the I-131 fibrinogen were eluted together with I-125 fibrin. We conclude that des-AB fibrin remains soluble in plasma at 20°C as well as at 37°C, forms aggregates or undergoes a conformational change increasing its molecular size and does not form complexes with fibrinogen at 37°C (Supported by the Deutsche Forschungsgemeinschaft)
Acquired Factor X (Stuart Factor) Deficiency in a Patient with Mycoplasma Pneumonial Infection
Authors: Peuscher F.W. , van Aken W.G. , Swaak A.J.G. , Sie L.H. , Statius van Eps L.W.
Published: May 2019

An isolated deficiency of factor X is known to occur in a hereditary form, the Stuart-Prower disease, and in an acquired form in some patients with para-proteinaemia and sporadically in systemic amyloidosis. Transient deficiency of factor X In the presence of normal levels of factors II, VII and V appears to be rare. In the literature, only three cases have been described. We have studied a patient with a severe haemorrhage diathesis and concomitant mycoplasma pneumonlal infection. The bleeding tendency proved to be due to isolated factor X deficiency. No circulating inhibitors of factor X were present. Systemic amyloidosis could not be demonstrated. Factor X-related antigen could not be detected (this test was kindly performed by Dr. Daryl S. Fair, Scripps Clinic and Research Foundation, La Jolla, U.S.A.). Treatment with vitamin K, prothrombin complex concentrate, fresi plasma and whole blood proved not to influence factor X activity in the patient’s Plasma. However, 20 days after admission·to hospital both factor X activity and antigen spontaneously returned to normal. These results suggest that the synthesis of factor X was transiently defective. Since other conditions known to affect factor X activity could not be demonstrated, it is postulated that the acquired factor X deficiency in this patient was related to the infection with mycoplasma pneumoniae.
The Effects of Three Potential Antithromotic Agents on Platelet Activities Ex VNO.
Authors: Cockbill S. , Taylor Patricia M. , Heptinstall S.
Published: May 2019

The effects of ingested acetylsalicylie acid (ASA), flurbiprofen (FN) and indemethacin (IN) on the platelet release reaction (ADP-induced release of 3H-serotonin from platelets) and on prostaglandin synthetase activity (PGSA; conversion of arachidonic acid to malon-dialdehyde by platelets) were determined. The studies were carried out on platelets from volunteers who had ingested a single dose of ASA (300 mg), FN (50 mg) or IN (25 mg) or were taking ASA (1x300 mg), FN (4x50 mg) or DJ (4x25 mg) daily. Two hours after a single dose of ASA both the release reaction and PGSA were completely inhibited. Both activities slowly returned in parallel over several days. During the continuous administration of ASA both activities were completely abolished until the ASA was discontinued. The effects of FN and IN on platelet activity were similar to each other but different from those of ASA. Two hours after a single dose the release reaction was abolished but PGSA was only partially inhibited. An effect on the release reaction could still be observed after 24 hours viiereas PGSA had returned to normal. During the continuous administration of FN and DJ, PGSA remained steady at about 15% and 50% of normal respectively, returning to normal 24 hours after the drugs were discontinued. Since FN and IN inhibit release without a long-lasting effect on PGSA they should be investigated further as potential antithrombotic agents.
The Effect of Antiplatelet Drugs on Piasma Retatiromboglobulin in Coronary Artery Disease
Authors: Han P. , Turnie A.G.G. , Genton F. , Gent M.
Published: May 2019

Platelets play a role in the development and complications of coronary artery disease (CAD) and a number of abnormalities of Platelet function which can be corrected by antiplatelet drugs have been described. Betathromboglobulin (BTG), a platelet-specific protein which is released from α-granules during platelet activation is significantly elevated in patients with angiographically demonstrated CAD (51.0 ± 31.0 ng/ml., n = 50) compared to normal (28.0 ± 8.0 ng/ml., n = 70) p < 0.001 . The effect of sulphinpyra-zone (800 mg.) or aspirin (1200 mg.)/dipyridamole (200 mg.) on plasma BTG in CAD was studied in a blind prospective crossover trial in 25 patients, Mean BTG concentration pre-treatment was 52.3 ng/ml. and after 1 month’s treatment with placebo, sulphinpyra-zone or aspirin/dipyridamole mean plasma BTG concentrations were 53.5, 49.6 and 56.7 ng/ml. respectively. Analysis of variance showed no significant difference between the means (p > 0.1). This study confirms increased plasma BTG concentrations in patients with CAD and indicates that therapeutic doses of these antiplatelet drugs do not significantly effect the BTG level and thus appear not to prevent α-granule release in CAD.
Modification of the Outcome of Coronary Thrombo-Embolism by Low and High-Dose Aspirin
Authors: Moore S. , Belbeck L.W. , Pineau S.A.
Published: May 2019

Previous work in this laboratory has shown that an occlusive thrombus (O.T.) induced. the left anterior descending coronary artery (L.A.D.) of dogs, by the placement of a segment of magnesium-aluminum wire (M.A.W.) in the lumen of the artery, is associated with the development of full thickness infarct (F.T.I.). Incomplete (mural) thrombus is associated with focal areas of ischaemic damage, mainly sub-endocardial, in the myocardium of the antero-lateral left ventricle. A 1.0 cm. length of M.A.W., shaped in the form of a tennis racquet, was placed by cardiac catheterization in the proximal L.A.D. of 30 mongrel dogs. Ten dogs received low dose aspirin (L.D.A.); (14 or 29 mgm/Kg daily in a single dose). Ten dogs received high dose aspirin (H.D.A.); (100 mgm/Kg daily in divided doses). Treatment was begun one day before operation and continued until death or killing. Ten control dogs received no drug. All control dogs died within 3 days of operation. Five had O.T. and F.T.I. The remainder showed focal ischaemic myocardial damage. None of the L.D.A. group died. At autopsy 3 or 4 days after operation, none had O.T. or F.T.I. In the H.D.A. group 3 died; 4 had O.T. and F.T.I. Thus L.D.A. provided significant protection from death, O.T., and F.T.I. H.D.A. provided some (P <0.005) but less protection from death but did not significantly affect the incidence of O.T. or F.T.I. Supported by Ontario Heart Foundation Grant T15-7.
Increase of Fibrinopeptide a After Concentrates of the Prothrombin Complex
Authors: Viganò S. , Cattaneo M. , Mannucci P.M.
Published: May 2019

Prothrombin complex concentrates (PCC) are known to carry a risk of thromboembolism. Platelet count, fibrinogen. FDP and other tests indicative of intravascular activation of the clotting mechanism are usually unchanged following PCC administration in patients The problem of detecting early signs of activation of the clotting mechanisms was thus reassessed by means of sensitive parameters of platelet damage and thrombin formation such as β-thromboglobulin (BTC) and fibrinopeptide A (FpA). FpA was significantly increased above baseline levels 30 to 90 min after the infusion of five types of commercial PCC to ten hemophilia B patients treated for fourteen episodes of spontaneous bleeding A more marked FpA increase ensued the infusion of the activated PCC FE IBA and Auto IX in seven hemophilia A patients with FVIII inhibitors treated on nineteen occasions There was no change after FVIII concentrates administred to a control group of five he mophilia A patients. Changes of BTG were observed more rarely; FDP, fibrinogen, platelet count, antithrombin III and paracoagulation tests were not modified These find: suggest that formation of thrombin in the circulation is likely to be a frequent event after PCC administration, though clinical manifestations of thromboembolism appear to be relatively rare.
Intermediate Purity Factor VIII by the Thaw-Siphon Cryoprecipitate Procedure
Authors: Mason E.C. , Pepper D.S.
Published: May 2019

The procedure begins with the separation of a 220 ml plasma donation into a plastic pack fitted with an internal siphon tube, which is an extension of the external tube connected to the satellite pack. The tube is arranged in an L-shaped configuration with one section aligned along the side edge of the pack, and the continuation along the bottom edge of the pack; the latter has perforations at regular intervals.

The donation is frozen as a flat slab at -40°C, and is, subsequently, thawed in an upright position in a 3–5°C water bath, with continual siphoning of thawed phase into the attached satellite pack. This proceeds for 40-50 minutes, when the frozen mass has reduced to a matrix, 5-10 ml in volume, of essentially pure ice trapping the cryoprecipitate. Citrate saline is introduced to dissolve the cryoprecipitate.

The procedure has been characterized by measurements of conductivity and protein, in eluant fractions, whilst investigation of the product for Factor VIIIC, total protein and fibrinogen, give respectively 150 u, 350 mg and 125 mg, i.e. 0.43u/mg protein.
Inhbition of Platelet Thrombus Formation by Chlorpromazine Acting to Diminish Haemodynamically Induced Haemolysis
Authors: Born G.V.R. , Uehmeier A.
Published: May 2019

The idea that drugs capable of counter-acting hypotonic haemolysis (Seeman, 1972, Pharmacol Rev., 24, 583) might diminish the activating effect of erythrocytes on platelets (Gaarder et al., 1961, Nature, Lond., 192, 531) was suggested by one of us (GVRB) as a novel approach towards inhibiting their intravascular aggregation as thrombi. Indeed, chlorpromazine added to human blood in concentrations which diminish haemolysis but have no direct effect on platelet aggregation (Mills and Roberts, 1967, Nature, Lond., 213, 35) prolong the “bleeding time” from small holes in artificial vessels where extravasation is terminated, as in living arterioles, by aggregated platelets (Born, Bergquist and Arfors, 1976, Nature, Lond., 259, 235). Apyrase had a similar effect, suggesting that it was due to decreased plasma ADP. We now provide evidence that this ADP is released by haemolysis which is diminished by chlorpromazine. Human venous citrated blood at 37° was pumped continuously through polyethylene tubing 280 μm internal diameter. Chlorpromazine caused concentration-related increases in “bleeding times” from a standard cut and decreases in free haemoglobin. The observed haemolysis would provide enough ADP to initiate platelet aggregation. The results support the suggestion (Born et al., 1976) that drugs with this effect of chlorpromazine may prevent haemorrhage-induced, eg. coronary thrombosis.
Comparison of Platelet and Plasma Fibrinogen from a Subject with the Congenital Abnormality, Fibrinogen Paris I.
Authors: Jandrot-Perrus M. , Mosesson M.W. , Denninger M.H. , Mênaché D.
Published: May 2019

Fibrinogen (ϕ) was obtained from normal platelets and plasma and compared with that from a subject with the congenital abnormality termed ϕ Paris I. The abnormality in this plasma ϕ is characterized by the presence of relatively large mutant γ chains (γ Paris I) that replace 50% or more of the normal γ chain population and unlike γ chains do not form covalently linked dimers in the presence of f. Xllla. Extracts of washed or washed trypsin-treated platelets, lysed in the presence of 8 M urea, were subjected to CM-cellulose chromatography. The ϕ containing peak from each sample was then concentrated and analyzed by SDS gel electrophoresis. All unreduced samples (5% acrylamide) possessed a ϕ band migrating in the same position as intact ϕ. ϕ-containing slices from duplicate unstained gels of platelet samples were reduced with DTT and again subjected to SDS electrophoresis (10% acrylamide). All samples showed bands corresponding to the Aα, Bβ and γ chains of normal plasma ϕ; the band corresponding to the γ Paris I chain was not observed. Furthermore, there was no evidence of the presence of γ Paris I chains in reduced specimens of a cross-linked fibrin clot from the γ Paris I subject’s platelets. The fact that γ Paris I chains are absent from platelet ϕ Paris I subject, supports the conclusion that platelet and hepatic ϕ pools are separate pools and suggests that platelet and hepatic ϕ are not assembled from the same gene product.
Correction of the Delayed Fibrin Aggregation of Fetal Fibrinogen by Partial Removal of Sialic Acid
Authors: Galanakis D.K. , Mosesson M.W.
Published: May 2019

Human umbilical oord fibrinogen characteristically displays delayed fibrin aggregation under conditions of relatively high ionic strength. This delay is greater in fibrinogen obtained from premature (p) (24–35 weeks gestation) infants, as compared with that from full term (FT) infants. We compared the sialic acid content of (fraction I-2) fetal (P and FT) with adult (A) fibrinogen, obtained from pooled plasma. The mean sialic acid μg/100mg protein) values were: P, 818 (±135 SD, range 621–1030); FT, 720 (±212, range 505–1280); A, 626 (±110, range 487-806). One P fibrinogen preparation (thrombin time 22.6 seconds) was partially desialated (resulting sialic acid value 490) by incubation with Vibrio cholera neuraminidase, dialyzed , and precipitated with ethanol to remove free sialic acid. The thrombin time of the resulting preparation was 15.4 (range 15.2–15.8), compared to 16.1 (range 15.5–16.4) for untreated A fibrinogen. The results suggest that the delayed fibrin aggregation of fetal fibrinogen is attributable to its relatively high sialic acid content. Moreover, the intermediate sialic acid (and thrombin time) values of FT (as compared to those of p) fibrinogen intimate the presence of a mixture of adult and fetal fibrinogen in full term cord blood.
Possible Role of a Membrane Phosphopolypeptide in the Inhibition of Platelet Function by Cyclic Amp
Authors: Fox J.E.B. , Smith A. , Haslam R.J.
Published: May 2019

The inhibition of platelet function by PGE1, which is mediated by cyclic AMP, is associated with an increased phosphorylation of a 24,000 dalton platelet polypeptide (P24) (Haslam et al. (1979) Biochem J. 178, 397). The significance of this phosphorylation reaction has been investigated. Washed human platelets that had been labelled with 32P were I incubated with 2 μM PGE1 for 2min, sonicated and separated into subcellular fractions by differential centrifugation. Analysis of the phosphopolypeptides present in these fractions by SDS-polyacrylamide gel electrophoresis showed that P24 was enriched in a 19,000- 90,000 g fraction that contained both plasma and intracellular membranes and accumulated 45Ca2+ by an ATP-dependent, oxalate-stimulated process. Uptake of 45Ca2+ by membranes from PGE1-treated platelets was significantly greater (approx. 50%) than by membranes from control platelets. When the former membranes were loaded with 45Ca2+ in the presence of oxalate and centrifuged througll a discontinuous sucrose density gradient, thre membrane fractions were obtained. Enrichment of both 45Ca2+ and P24 was greatest in the most dense of these. Since platelet responses to aggregating agents are believed to be mediated by Ca2+ ions, we suggest that PGE1 may inhibit these processes by causing the cyclic AMP-dependent phosphorylation of the membrane-bound P24 which then stimulates the active transport of Ca2+ ions out of the platelet cytosol.