bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Factor VIII-Inhibitor By-Passing Activity (FEIBA)
Authors: Hess K. , Shih N. , Tishkoff G.
Published: May 2019

In an attempt to identify the thrombogenic factor in human factor IX concentrates, we have studied the role of trace quantities of activated clotting factors employing an assay that compares the Factor VIII-like activity of IX concentrates with the ability of these products to restore to normal the abnormal activated partial thromboplastin time (APTT) of Factor VIII inhibitor plasma after 1 minute and 40 minute incubation. A coagulant activity (FEIBA) was evident when partially purified Factors X and II were combined in vitro. Factor Xa (4 × 10-4 u) plus Factor II gave negative results. Factor IIa (5.5 × 10-2u), when combined with Factor X, generated FEIBA. Activated clotting factors (Xa, IIa) when tested alone, at comparable levels, were devoid of FEIBA. Our results suggest a mechanism, distinct from activated clotting factors, that can effectively by-pass the Factor VIII defect in the coagulation cascade. The proposed mechanism appears to also by-pass the normal inhibitory properties (i.e., antithrombin III) of human blood.
Distinctive Neoantigenic Marker Associated with a Plasmic Derivative (Fragment D-D) of Cross Linked Human Fibrin
Authors: Budzynski A.Z. , Marder V.J. , Parker M.E. , Shames P.
Published: May 2019

Fragment D-D is a plasmic degradation product of crosslinked fibrin consisting of 2 Fragment D molecules linked by Factor XIII-induced ε(γ-glutamyl)lysine bonds. In this study, the antigenic markers of Fragment D-D were compared with those of Fragment D obtained from fibrinogen and non-crosslinked fibrin, as well as with those of fibrinogen Fragments X, Y and E. An antiserum against Fragment D-D raised in chickens and absorbed with fibrinogen and Fragment D showed immunoprecipitation with Fragment D-D but not with fibrinogen or with the other derivatives. In a double antibody radioimmunoassay system the antiserum bound 125-I labeled Fragment D-D but not labeled fibrinogen or Fragment D from fibrinogen and non-crosslinked fibrin. The binding of labeled Fragment D-D was inhibited by non-labeled Fragment D-D at concentrations of 10-10,000 ng/ml and was equally reliable in a plasma milieu. Purified plasmic degradation products of human fibrinogen inhibited binding less effectively. The increased assay sensitivity for Fragment D-D appears to be related to specific neoantigenic sites associated with the overall structure of this degradation product and provides molecular basis for the differentiation between fibrinogenolysis and thrombolysis.
The Antithrombotic Potential of Dihomo-γ-Linolenic Acid
Authors: Kernoff P.B.A. , Davies J.A. , McNicol G.P. , Willis A.L. , Stone K.J.
Published: May 2019

Dihomo-γ-linolenic acid (DHLA; Ro 12–1989, Roche), a natural precursor of prostaglandin E1, has been evaluated as a potential antithrombotic agent by studying its effects on platelet function, blood coagulation and blood viscosity. In vitro investigations were performed on blood obtained from 10 normal subjects and 10 patients with diabetes mellitus, 8 of whom had evidence of severe microangiopathy. Prior incubation of normal platelets with DHLA caused a marked and dose-related inhibition of : platelet aggregation in response to stimulation with ADP, collagen, epinephrine, thrombin and ristocetin; platelet glass-bead retention; release of serotonin and platelet factors 3 and 4. Effects on diabetic platelets were less marked but still highly significant. DHLA also reversed platelet aggregation and inhibited the adhesion of porcine platelets to collagen-coated glass. No significant effects were found on whole-blood viscosity or tests of blood coagulation apart from a slight shortening of the one-stage prothrombin time. Ex vivo studies were performed on 3 normal volunteers after oral administration of single doses of DHLA. Increased plasma DHLA/arachidonic acid ratios resulted in all instances (Stone et al, this meeting). No adverse effects were noted but consistent evidence of a favourable change in haemostatic tests was not obtained. The spectrum of in vitro inhibitory activity of DHLA on platelets is wider than that reported for other potential anti-thrombotic agents and our results suggest that DHLA shows considerable promise in this respect.
The Influence of Cross-Linking and Plasminogen on Kinetics of Fibrinolysis
Authors: Whitaker A.N. , Gaffney P.G.
Published: May 2019

The influence of cross-linking upon the kinetics of fibrinolysis has been studied in an in vitro system by monitoring the release of radioactive label, peptide materials, fragment X and D-dimer. Cross-linked (XL) and non-cross-linked (NXL) fibrin clots were prepared by clotting citrated plasma containing 125-I labelled fibrinogen with human thrombin, in the presence respectively of 40 mM Ca C12 or 2 mM EDTA, and incubating for 2 hours at 37°C. After washing, clots were placed in buffer containing human plasminogen (glu- or lys-) in concentrations ranging from 0 to 10 caseinolytic units per ml. Clots were transferred after 30 minutes to a solution of streptokinase (1000 units per ml) and the supernatants subsampled serially for 3 hours. NXL fibrin lysed progressively and sometimes completely. Maximal lysis rates were achieved with intermediate concentrations of plasminogen. XL fibrin lysed slowly and significant lysis was obtained only after exposure to the higher concentrations of lys-plasminogen. XL-fibrin lysed more readily after exposure to glu-plasminogen than to lys-plasminogen. Analysis of the chains in XL fibrin clots by SDS-polyacrylamide electrophoresis revealed the presence of a small residuum of NXL fibrin and incomplete cross-linkage of α chains. The NXL component lysed preferentially in streptokinase and the terminal clots contained only XL fibrin. Parallel effects may operate in vivo. The data support the contention that fibrinolytic mechanisms readily deal with NXL fibrin, and that only fibrin which has been XL occurs in thrombi and is of pathologic significance.
Isolation of Platelet Specific Glycoprotein Antigen
Authors: Kosaki G. , Ohara S. , Imaoka S.
Published: May 2019

Hanna and Nelken in 1971 observed two specific antigens on human platelets. One is a glycoprotein, m.w. 110,000, with α-electrophoretic mobility and the other is protein, m.w. 30,000. We have isolated a platelet specific glycoprotein antigen m.w. 70,000, with β-globulin mobility. Differentially centrifuged and Sepharose 2B gel filtered platelets were suspended in deionized water, frozen, thawed and sonicated. One volume of 1.2 M perchloric acid (PCA) was added and mixture centrifuged and the supernatant dialysed against water and lyophilized. Antiserum was obtained by s.c. injection of this extract to a rabbit. After absorbtion with 0.6M PCA extract of human plasma and erythrocytes, this antiserum showed one precipitin line with platelet PCA extract, but no line with PCA extract of plasma, erythrocyte or lymphocyte on double diffusion. Furthermore, it formed no precipitin line with pus, liver, colon, stomach or kidney, but a weak line with lung and a strong line with spleen which fused with the platelet line. By gel filtration of crude platelet PCA extract on Sephadex G-200, antigenic activity was observed in the third fraction. On 7.5% Polyacrylamide disc electrophoresis of this fraction, three bands stained with amidoblack and PAS. Main band was the middle one of m.w. 70,000 with β-globulin mobility. Positive immunofluorescent staining was found on platelets of human buffy coat smear, but not on erythrocytes or lymphocytes. The absorbed antiserum showed precipitin line with serum from whole blood and recalcified platelet rich plasma, but not with serum from recalcified platelet poor plasma. β-thromboglobulin is released from platelets during coagulation. We are investigating the immunological identity of β-thromboglobulin with our antigen. The result will be discussed.
Venous Doppler Velocity Examination in Pulmonary Thromboembolism
Authors: Griggs T.R. , Bustad L.B. , Ely C.A.
Published: May 2019

The Doppler ultrasonic velocity detector, which is sensitive for deep vein thrombosis in 90% of cases demonstrated by venography in our institution, might be useful in the evaluation and management of patients with pulmonary thromboembolism (PE). This study describes the association of results of Doppler velocity examinations with the presence or absence of PE as documented by pulmonary angiography in 51 patients. Results are shown in the table. Doppler studies were performed within 48 hr of angiography.

Five patients with positive angiograms had initial or recurrent PE while on anticoagulants. Four of the 5 had positive Doppler studies and the fifth had an incomplete test because of a cast. One patient developed Doppler positivity 2 days before reembolization.

One conclusion is clear: Negative venous Doppler examinations must not be used to rule out PE. The study also raises 2 questions:1) Does negative Doppler examination at the time of PE represent complete embolization of thrombus and low risk of immediate recurrence? 2) Is positive Doppler study at the time of PE an indication for early venous plication?
Estimation of Thrombus Formation by Isotopic Methods and by Gravimetry in Experimental Thrombosis
Authors: Zimmermann R. , Lange D. , Weckesser G. , Zeltsch C.
Published: May 2019

Composition of venous and arterial thrombi differs from that of blood and may change in different thrombogenic conditions and in antithrombotic therapy. In 130 rabbits thrombus formation was investigated bv labeling of platelets (51Cr), fibrinogen (125J) and red cells (59Fe or 51Cr). Thrombosis was induced in 3 different models:I. Formation of slow growing arterial and venous thrombi, II.rapid venous thrombus formation within 20 min by application of silver nitrate and III.production of stasis thrombi. The' radiometric thrombus size’ was related to the thrombus weight by means of Spearman rank correlation. The' specific thrombus/blood ratio’(cpm g thrombus/cpm g blood) was measured in the series I in venous and arterial thrombi to be 19.1 and 50.9 (platelets), 12.6 and 6.1 (fibrinogen) and 5.1 and 1.7 (red cells) and in the series II 5.7 (fibrinogen) and 1.5 (red cells). III. In stasis thrombi these values resembled nearly to that of blood. Dependent on the pathogenetical mechanism of thrombus formation, a different incorporation of blood elements could be stated with the greatest discrepancies in the uptake rates of red cells in arterial thrombi. Statistical analysis of methods used to measure thrombus size demonstrated only in the venous.system a reliability for measuring thrombus size by isotopic methods. Correlation coefficient was 0.8 (51Cr), 0.76 (125J) and 0.83 (59Fe) in the model I and 0.84 (125J) and 0.92 (51Cr red cells) in the model II.
Separate Mechanisms for the Inhibition of Platelet Aggregation by Adenosine and 2-Cloroadenosine
Authors: Apitz-Castro R. , Torres C.R.
Published: May 2019

The mechanism by which adenosine (Ado) and 2-cloroadenosine (Cl-Ado) inhibit platelet aggregation is not clear. In order to get some insight into the mode of action of these compounds, we studied the effect of Cl-Ado on the uptake of Ado by intact platelets, the effect of these compounds on the endogenous phosphorylation of specific plasma membrane proteins, and its effect on the carboxymethylation pattern of plasma membrane proteins in intact platelets. Cl-Ado does not modify the uptake of Ado by intact platelets, nor is itself incorporated into the platelet’s pool of nucleotides. Phosphorylation of plasma membrane proteins is not affected by Cl-Ado; however, Ado produces a selective increase in the phosphorylation of one plasma membrane component of glycoproteic nature. As has been reported, phosphorylation of this glycoprotein is also modulated by cAMP (BBA, 455:371, 1976). Although the electrophoretic pattern of carboxymethylated plasma membranes is unaffected by Ado or Cl-Ado, it was found that the former markedly increases the label of all the susceptible proteins, while Cl-Ado selectively protects a single membrane component. Electrophoretically, this component seems to be related to the above mentioned glycoprotein. The results reported suggest that Ado and Cl-Ado interact with different components of the plasma membrane, impairing platelet aggregation through different mechanisms. In the case of Ado, two ways seem operative: a) A cAMP-like stimulation of a specific membrane glycoprotein and b) A more general perturbation of the membrane structure, perhaps through an Ado-carrier complex (Acta Med. Scand. 525:169, 1971). Cl-Ado seems to interact solely on the external surface of the plasma membrane, suggesting that the transmembrane phospho-glycoprotein previously described is in some way closely related to the ADP-receptor of the platelet plasma membrane.
Platelet Volume Distribution in Primary and Secondary Thrombocytopenias
Authors: Le Tohic F. , Le Menn R. , Boisseau M. , Samama M.
Published: May 2019

Platelet volume distribution was studied using a Coulter Z Bic channelyser model C 1000 in 28 idiopathic thrombopenic purpura [I.T.P.] and in 59 thrombocytopenias attributed to a failure of production. In 40 cases platelet volume distribution had an abnormal pattern with a first peak due to particles smaller than 3 N3. This abnormal curve was encountered in both groups of thrombocytopenias and its frequency was in good correlation with the degree of thrombocytopenia. In 24 out of these 40 cases the albumin gradient separation gives a platelet suspension the volume distribution curve of which has a normal pattern In 16 other cases (6 I.T.P. + 10 secondary thrombocytopenias) it was not possible to supress the interference of the smaller particles. An increase of the mean platelet volume and of the Karpatkin index (% of platelets > 12 N3) was present in 18 out of 22 I.T.P. but was also occasionally found in secondary thrombocytopenias. Electron microscopic and cytoenzymo1ogic studies were used to identify the smaller particles of about 1 N diameter and to determine the relative proportion of red cells and platelets fragments. It is also important to investigate if these particles are present in circulating blood or are formed after blood collection.
Isopycnicyty and Relations Between Platelet Volume, Density and Age
Authors: Boneu B. , Caranobe C. , Vigoni F. , Blasco A. , Bierme R.
Published: May 2019

The conflicting results, published in literature, concerning the correlation and significance of platelet volume, density and age are possibly due to the different experimental procedures utilized : medium, densities or centrifugation. We present here the results obtained in man with two methods of centrifugation (I : 800g/60mn ; II : 7000g/20mn) and three different linear isoosmolar gradients (a : Ludox-PVP ; b : Albumin ; c : Metrizamide).

With the methods I a, b, c we obtain only one gaussian platelet population. There is an evident relationship between apparent platelet densities and their volumes : the denser the platelets the larger they are. The platelets of each fraction present a log normal volume distribution. The platelet survival studies (51 Cr) demonstrate that platelet volume and age are independent. With the methods II a, b, c we also obtain only one gaussian platelet population but no significant variation of volume between dense and light platelets (except for Ludox-PVP). The mathematical analysis of platelet kinetics (75 Se - 51 Cr) indicates : (1) that young platelets (age < 48h) present the same heterogeneity as the whole platelet population, but the mean density is lower ; (2) the potential survival of platelets of various densities is identical ; (3) the young platelets increase slightly their density during the first four days of their survival. Thus there is continuously some sort of exchange between platelet populations, (peripheral maturation phenomenon).

In conclusion ours are conflicting results too. We demonstrate in fact that the second method achieves an isopycnic separation but not the first one.
The Relationship Among Platelet Age, Volume and Electrophoretic Mobility
Authors: Motomiya T. , Yamazaki H. , Sawasaki Y. , Nakajima O.
Published: May 2019

The platelet carries a net negative charge and its change may be an important factor in platelet adhesion and aggregation. Platelet age and volume are also believed to be important factors in its functions. Six male rabbits were injected with 50 μCi of 75Selenomethionine intravenously and EDTA-blood was drawn 3 days after. Platelets were washed 3 times and suspended in 3 mM triethanolamine acetate carrier buffer. Platelet electrophoresis was performed using with a Continuous Free-Flow Electrophoresis VAP-5 (Bender & Hobein, Munich). Electrode buffer used was 100 mM triethanolamine acetate buffer pH 7.4 and electrophoresis was performed at 17.5°C, 1250V and 80mA. Approximately 5×108 platelets were electrophoresed per hour into 90 fractions. The fraction #1 is the anode side and #90 cathode side. Platelets in each fraction obtained as a suspension in the buffer were studied for platelet count and volume determinations using with a Coulter Counter ZbI and a Channelyzer C-1000. The radioactivity was measured by a well-type scintilation counter. Platelets were separated well making a single peak at the 27±3.4 fraction. The radioactivity of platelet increased as the fraction number increased but platelet in bilateral side fractions was too small in number and determination of the radioactivity was difficult and the peak radioactivity was at the measurable extreme right fraction (68±3.8). The largest platelet volume also appeared at the measurable extreme right (cathode side) fraction. In conclusion, Continuous Free-Flow Electrophoresis separated platelets well by the difference of cell surface potentials. 75Selenomethionine labeled platelets which have been thought to be young seem to have low electrophoretic mobility and relatively large volume.
A Simple, Quantitative, Objective Measurement of Platelet Shape
Authors: Holme S. , Murphy S.
Published: May 2019

Previous studies of platelet aggregation have shown that platelet disc-to-sphere transformation induced by ADP is accompanied by a decrease in light transmission. Our studies were directed towards an understanding of this effect; they also resulted in the development of a method which quantitatively reflected platelet shape. A Payton aggregometer with a 0.5 ml cuvette was used. Electronic settings were standardized to give a read out of 100% transmission (10 mV) on the recorder with water in the cuvette, and 0% transmission (0 mV) with background current alone. For fresh, discoid platelet suspensions there was a fall in light transmission to a minimum at 200 r.p.m. with an increase in transmission to a maximum at 700 r.p.m. with increasing stirring speed After disc-to-sphere transformation by ADP, EDTA, or chilling, a constant light transmission, un-effected by stirring rate, was observed. When the changes in light transmission due to the disc-to-sphere transformation were measured, only a small decrease took place at 0 r.p.m. At 200 r.p. m. there was actually an increase due to the shape change, while maximum decrease was obtained at 700 r.p.m. This shows that the platelet shape change observed in the aggregometer is mainly determined by the difference of discoid and spherical platelets in their effect on light transmission in presence of stirring. When the ratio O.D.700/O.D.200 was determined for 38 suspensions of fresh normal PRP kept at 37°C, with platelet count ranging from 0.05 - 0.4 × 106 per mm3, there was little variation (0.66± 0.04 S.D.). The ratio approached unity with spherical platelets. With spherical and discoid platelets mixed in various ratios O.D.700/0.D.200 could detect as little as 10% change in discoid platelets, demonstrating that this ratio gives a sensitive, objective and quantitative reflection of platelet shape.
Morphological Sequence of Spontaneously Forming Parietal Thrombi as Observed by Scanning Electron Microscopy (SEM)
Authors: Genz H.J. , Metzger H. , Tauber P.F. , Ludwig H.
Published: May 2019

Spontaneous thrombus formation in human mesenteric veins was studied with the SEM. Tissue specimens were prepared according to Ludwig et al., Acta anatomica, 96, 469-477(1976). Platelet shape change, thrombus formation and organization and the morphological interactions between the various corpuscular elements of blood are demonstrated. The following morphological criteria of these processes are observed :(1) Platelets adhere to distinctly altered endothelial surfaces and exhibit pores in the membrane and pseudopodia. (2) Platelet aggregation and thrombus formation occur next to each other along the endothelial surface. Thrombi contain red blood cells and also a larger number of lymphocytes, but only a few platelets are hold prisoners within the fibrin network. Once caught in the mesh, such platelets do not show shape change compared to those being in contact with the endothelium. (3) Red blood cells between the thrombus fibers undergo form changes. Lymphocytes remain unaltered, but vice versa destroy adjacent fibrin fibers leading to partial loss of thrombus stability. This destruction occurs to a much lesser degree when platelets are near to the lymphocytes. It seems conceivable that platelets exert an inhibitory effect towards lymphocyte-induced fibrin proteolysis. The data suggest that both platelets and lymphocytes possibly represent a cellular control system that is responsible for the physiological clearance of spontaneously formed thrombi.
Typical Changes of Platelet Volume During and After Extracorporeal Circulation
Authors: Wenzel E. , Saavedra E. , Volkmar I. , Monadjemi H. , Pöhler E. , Stapenhorst K. , Harbauer G. , Isringhaus H.
Published: May 2019

In 32 patients undergoing open-heart-surgery with extracorporeal circulation (ECC), platelet volume distribution curves (PVD) during and immediately and 24 and 48 hours after cardiopulmonary bypass were examined by an electronic particle size analyzer based on the Coulter-Counter-System. The PVD changes, i.e., the reduction of the mean platelet volume (MPV) and the platelet fall correlates significantly with the bypass time (r = 0.78). Two hours after the end of ECC, these changes remained completely irreversible or only partially reversible in patients with perfusion times longer than 60 minutes. In most of the patients with shorter perfusion times, we observed a normalisation of PVD curves and return of platelets to counts approaching normal levels.

Some plasma coagulation parameters (i.e., concentration of clottable fibrinogen, FDP) were estimated in parallel and were found to be in good correlation with the platelet defects.

In patients with marked changes of MPV up to 48 hours after the end of surgery, a pathologic volume distribution curve of thrombocytes was observed, but platelet counts returned in most of them to normal values. This indicates that the determination of PVD, as a simple reliable diagnostic parameter, gives more essential information about platelet damage than does the platelet count alone.
Purification of Human Factor
Authors: Bolhuis V.P.A. , Hakvoort T.B.M. , Breederveld K. , Mochtar I.A. , ten Cate J.W.
Published: May 2019

Human factor V has been purified from cryo-supernatant by fractionated precipitation with polyethylene glycol (6000, 16-24% w/v, yield 65%), followed by gelfiltration on AcA 44 in Michaelis buffer with 50mM Ca++ (elution in Vo, yield 90%), adsorption of contaminating haptoglobinpolymers on hemoglobin bound to Sepharose-4B (yield 95%) and adsorption of the glycoproteins (mainly factor V) onto concanavaline A bound to Sepharose-4B, elution with α-methylpyranoside (4M) and gelfiltration of the factor V eluted. The purified factor V hasa specific activity of about 28 u/mg and thetotal yield is 16%. The native protein and the subunitsobtained by reduction in the presence of dodecyl sulphate and urea have been characterized by Polyacrylamide gel electrophoresis.
A Simplified Method for Preparation of Highly Purified Human Thrombin
Authors: Seghatchian M.J. , Miller-Andersson M. , Gaffney P.
Published: May 2019

Increasing use of synthetic substrates for potency estimation of biological activities within the coagulation field has raised a demand for a supply of highly purified thrombin. We have simplified a thrombin preparation procedure using clinical factor IX concentrates as starting material. Activation was performed using fresh brain thromboplastin prepared in a simplified way. A first ionic exchange chromatography step was used, which got rid of 80% of the impurities. The thrombin was further purified on SP Sephadex comparatively following two different pathways. In one case the whole purification procedure was performed at pH 6.5 which led to a thrombin with proposed high stability but which gives a relatively low-yield preparation. In another purification procedure the preparation was performed at pH 7.4 which gives a considerably better yield but possibly poorer stability upon storage. In both cases a final purification step on heparin gel was performed. The pH 6.5 thrombin could be separated into two fractions, one with a specific activity of 2500 iu/mg and another with a specific activity of 6000 iu/mg. A protein peak corresponding to the highly active thrombin was obtained also from the pH 7.4 thrombin, but that material had lost its biological activity. The different thrombin preparations have been carefully ampouled and sealed under nitrogen in order to faciliate further stability studies and comparative studies on bio-assays using clotting methods as well as synthetic substrate methods.
New Chromogenic Peptide Substrate for Factor Xa
Authors: Aurell L. , Simonsson R. , Arielly S. , Karlsson G. , Friberger P. , Claeson G.
Published: May 2019

The chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA is based on the primary structure preceding the bonds split by factor Xa in bovine prothrombin. Substitution of amino acids in this natural sequence by closely related amino acids has only given inferior substrates. Thus, the natural sequence seems very important. However, we have found that the free γ-carboxyl group of Glu is not indispensable. Substrates with the above structure but having been derivatized on the γ-carboxyl group of Glu, in the form of simple esters or amides, show improved properties. Especially Km of the new substrates compare favourably with our first substrate. Some of the amides also show increased Vmax.

These improved properties have made it possible to increase sensitivity, shorten incubation times and lower substrate consumption in several methods where this type of substrate is utilized.
Human Prothrombin/Phospholipids Interaction. Competitive Effect of an Anticoagulant Venom Phospholipase A2
Authors: Boffa G.A. , Benarous R. , Boffa M.C.
Published: May 2019

The high anticoagulant activity of the phospholipase A2 isolated from a Viperidae venom was explained by the formation of a complex with phospholipid at its protein binding site (1). The anticoagulant effect was reversible with specific antiphospholipase A2 immunoglobulins.

The interaction between 125 I prothrombin and phospholipid in the presence of calcium was impaired by the phospholipase. A competitive effect between prothrombin and the phospholipase was demonstrated. It did not appear sufficient to explain the anticoagulant property of the enzyme. This effect was abolished after photo-oxidation of the phospholipase. In the presence of another Viperidae phospholipase A2, devoid of anticoagulant activity, no competitive effect was observed.

The presence of several sites responsible for anticoagulant, catalytic and recognition properties have been looked for by various partial chemical denaturation processes.
Preparation of a Nonthrombogenic Prothrombin Complex Concentrate
Authors: Chandra Sudhish , Wickerhauser Milan
Published: May 2019

The major problem faced in the preparation of prothrombin complex concentrates (PTC)for clinical use is the activation of clotting factors. This activation may cause thrombo-embolic complications when such a preparation is administered to the patients with liver impairment or factor IX deficiency. We have developed a large scale procedure which minimizes this problem and leads to a substantially safer preparation. Plasma supernatant, after recovery of cryoprecipitate, is adsorbed with kaolin to remove contact factors. Kaolin supernatant is fractionated by precipitation with 10% polyethylene glycol 4000 (PEG) to remove fibrinogen, plasminogen and factors that might contribute to the undesirable activation of PTC. PTC is then extracted from the 10% PEG supernatant by batchwise adsorption and elution with DEAE cellulose. The eluting buffer is enriched with a small amount of antithrombin III to maintain the stability of the product against possible activation. The eluted PTC is concentrated by lyophilization, desalted on a Sephadex G-50 column, sterilized and lyophilized under aseptic conditions. The final concentrate was 70-fold purified and the recovery was 30% over starting plasma in terms of factor IX activity. The preparation was nonthrombogenic in vitro by the nonactivated partial thromboplastin time test and in vivo by the stasis model (Wessler et al J. Appl. Physiol. 14, 943, 1959) using 90 F IX units/Kg. body weight of the rabbit.
Turnover Studies of Des-A Fibrin and Fibrinogen in Healthy Human Beings
Authors: Müller-Berghaus G. , Mahn I.
Published: May 2019

Soluble fibrin was injected into man. Two methods were used to prepare soluble des-A fibrin. Purified human 1-125-fibrinogen was transformed into soluble des-A fibrin (des-A FM):(1) by thrombin in the presence of 2.0 M urea (method according to Hogg and Blombäck, 1975), (2) by ancrod. Eight male volunteers were injected either with thrombin-induced soluble I-125-fibrin (FM-T) or ancrod-induced soluble I-125-fibrin (FM-A). The control consisted of I-131-fibrinogen injected simultaneously with soluble I-125-fibrin.

The mean distribution volume of des-A FM-T was 48.9 ml/kg and that of des-A FM-A 50.3 ml/kg which is similar to that of the injected fibrinogen (43.0 ml/kg) indicating a homogenous distribution of the injected FM in the circulating blood. The injected des-A FM disappeared faster from the circulating blood than des-AB fibrin monomer. Six hours after injection 99.5% of the injected FM-A and 95% of the injected FM-T were removed from the circulating blood. The FM were cata-bolized since free I-125 were quantitatively recovered from the urine. The elimination curve of des-A FM-A as well as des-A FM-T does not represent a mono-phasic exponential decay.

The experiments demonstrate that des-A fibrin can be traced in man and is cleared from the circulating blood at a high elimination rate.

(Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg)
A Study of the Asphctic Newborn Related to Hypercoagulability by Determination of Soluble Fibrin Monomer Complex (SFMC) and Plasmin Inhibitor
Authors: Susuki S. , Graeff H. , Hafter R.
Published: May 2019

Gordblood samples from 80 healthy newborns and 19 asphyctic newborns were examined. SFMG and fibrinogen were precipitated from plasma with β-alanine.

Agarose gel filtration of redissolved precipitate resulted in separation of SFMC and fibrinogen. Other parameters such as TEG, Prothrombin time (PT), Partial thromboplastin time (PTT), and Plasmin-inhibitors (α2-macrogloblin, α1-antitrypsin, antithrombin III)were also determined. (Results)

(1) The percent amount of SFMC of total fibrinogen content in asphyctic newborn increased 4.73 ± 1.55%, while the remaining normal infants showed only 3.17 ± 0.55% (P < 0.05)

(2) In neither PT nor PTT can a significant difference be seen, although asphyctic newborn showed the tendency of hypercoagulability in TEG.

(3) α1-antitrypsin (92.8 ± 21.5)and antithrombin III(8.4 ± 2.3) levels were much lower in the group with asphyxia.

These results indicate that a low level of Plasmin-inhibitors act synergestically with a high activator value.

The low antithrombin III level in particular could be one of the reason for the development Hypercoagulability and DIC in asphyctic newborn infants.
In vivo effects of human granulocyte neutral proteases on blood coagulation in green monkeys (cercopithecus aetiops)
Authors: Egbring R. , Gramse M. , Heimburger N. , Havemann K.
Published: May 2019

Two neutral proteases (elastase-1ike = ELP, and chymotrypsin-like = CLP) derived from human PMN in highly purified form inactivate in vitro humanisolated coagulation factors (Thromb. Res. 6, 315, 1975). These effects can be observed also in human plasma, despite its high antiprotease capacity. (Blood February 1977).

ELP and CLP (both free of endotoxin) were infused into green monkeys either separate or in combination to investigate a possible role of these proteolytic enzymes also in vivo. Activity of coagulation factors I - XIII and antiprotease potential has been followed for a 24 hour period in short intervals.

A loss of activity of coagulation factor II, V, VIII, IX, X and XIII could be demonstrated. Fibrinogen concentration decreased and fibrinogen split products could be detected. In two-dimensional immunelectrophoresis α1-antitrypsin-ELP and α2-macroglobulin-ELP-complexes were demonstrable for about 6 hours after protease infusion. Bleeding complication occurred after injection of both enzymes.

The results indicate a direct proteolysis of coagulation factors by PMN neutral proteases in vivo.

We suggest that similar conditions are present in patients with acute leukemia or septicemia.
Vasoconstrictor and Vasodilator Agents in Disseminated Intravascular Coagulation
Authors: Whitaker A.N. , Bunce I.H. , Sleigh B.
Published: May 2019

Modification of disseminated intravascular coagulation by vasculoactive agents has been evaluated in rabbits infused with thrombin, measuring the extent of renal capillary thrombosis, suppression of creatinine clearance and elevation of plasma creatinine and pl. haemoglobin. Control studies included the administration of each agent without thrombin. The addition of angiotensin (0.1 μg/kg/min) consistently increased the extent of thrombosis and enhanced the other sequelae of thrombin infusion. Angiotensin was more potent than either norepinephrine or epinephrine (1 μg/kg/min). Intravenously, 5-hydroxytryptamine (1-10 μg/kg/min) decreased the severity of renal involvement and thrombosis. In contrast, if given intra-aortically 5-HT (1 yg per kg/min) enhanced all sequelae, being comparable to angiotensin. Propranolol, to blockade β-adrenoreceptors, did not significantly modify the response to thrombin, whether given alone or in addition to epinephrine. Blockade of α-adrenoreceptors with phenoxybenzamine variably decreased both renal lesions-and haemolysis produced by thrombin, and prevented the enhancement of these by epinephrine. Phenoxybenzamine blockade could be reversed by angiotensin. The addition of vasculoactive agents to thrombin only slightly influenced reductions in platelets and fibrinogen. While statistically significant, these small differences could not explain the differences in the pattern of thrombosis, α-adrenoreceptors in this model may be less critical than in the generalized Shwartzman reaction, but chemical mediators in general may be important in the expression of disseminated intravascular coagulation and thrombus localization.
Prevention of the Generalized Shwartzman Reaction by Bradykinin and Prostaglandins
Authors: Latour J.G. , Léger-Gauthier C. , Groulx C.
Published: May 2019

We have investigated, in the pregnant rat, the implications of Hageman factor and prekallikrein activation in the pathogenesis of the generalized Shwartzman reaction (GSR). We have found that unlike the hyperlipemia animal, the pregnant rat, which develops the GSR after a single injection of endotoxin (S. typhosa 0.4 mg) on day 20 of gestation, does not consume prekallikrein. Prekallikrein was estimated by measurement on TAME of the arginine esterase activity generated by kallikrein upon addition of active Hageman factor to the test plasma. Bradykinin triacetate (2 or 4 μg/kg/min.), prostaglandin (PG) E-, (0.3 μg/kg/min.), PGA2 (1.0 μg/kg/min.) or isoproterenol (0.66 μg/kg/min.) when given as a slow infusion over 4 hours, from the time of the endotoxin to the sacrifice of the animals, were found to prevent the GSR. On the other hand, aspirin (150 mg/kg) or propranolol (2 mg/kg) given before endotoxin enhance the incidence or severity of the GSR. Furthermore, aspirin markedly reduces the prevention generated by bradykinin infusion and to a less extent that of isoproterenol. Finally, indomethacin (7 mg/kg) given twice daily for 3 days sensitizes the male rat to the GSR, since a single injection of endotoxin triggers deposition of fibrin in the glomerular capillaries. It is concluded that bradykinin generates the release of PG’s and that together they prevent, like the α-adrenergic and the D-serotoninergic blocking agents and or like a β-adrenergic stimulation, the renal vasomotor reactions found necessary for the GSR. These results support the hypothesis that impairment of kinin generation may possibly account for the sensitization of the pregnant rat to the GSR.
Diffuse or Localized Intravascular Coagulation in Severe Cases of Malaria Tropica: Heparin or Not?
Authors: Vreeken J. , Goote Th. M.
Published: May 2019

The value of additional heparin therapy in patients with malaria tropica and signs of intravascular coagulation is far from clear. Whereas at one side heparin could perhaps be of value to prevent further obstruction of the microcirculation; at the other hand heparin could add additional bleeding risk when restoration of a hemostatic defect does not occur (e.g. when there is concomittant liver damage or uremic thrombopathy). Nine non immune patients with imported malaria tropica were followed during varying period for blood platelets number, coagulation factors, circulating fibrin monomers and fibrinogen degradation products (F.D. P./f. d. p. ). Three of them had severe renal damage and two signs of cerebral malaria. All patients showed initially before the 15th day of illness an episode of thrombocytopenia. None of them had signs of circulating fibrin monomers. Elevated levels of F. D. P. /f. d.p. were only occasionally found and were not seen in the patients with cerebral malaria and/or severe renal damage. Low fibrinogen levels were found in only three patients (all had renal damage). There were no signs of diffuse intravascular coagulation in those patients, some of the patients showed coagulation defects attributed to liver damage. Therefore intravenous heparin was not additionaly given. All patients recovered completely.
Platelet-Derived Growth Factor Production During the Platelet Release Reaction Independent of The Endoperoxide Pathway.
Authors: Ihnatowycz I.O. , Cazenave J-P. , Moore S. , Mustard J.F.
Published: May 2019

Ross et al (P.N.A.S. 71:1207, 1974) showed that a platelet-derived growth factor (PGF) promotes the proliferation of arterial smooth muscle cells (SMC) and thus may be important in atherogenesis. Although PGF is found in plasma when platelets are exposed to thrombin or collagen it has not been conclusively established that the material is released from platelet granules. Inhibitors of the release reaction and of platelet cyclo-oxygenase were used to examine the relation between release of 14C-serotonin (14C-5HT) from rabbit platelets and the appearance of PGF in the suspending fluid. Washed platelets (2 × 106/mm3) were suspended in Tyrode solution containing albumin and apyrase at 37°C and treated with collagen suspension, thrombin or ADP. After 5 min. the platelets were removed and the supernate assayed for 14C-5HT, malondialdehyde (MDA) and PGF. Incorporation of 3H-thymidine into cultured rabbit aorta SMC DNA was used as an index of PGF concentration. There was a direct relation between the concentration of collagen or thrombin and the amount of PGF in the supernate. Indomethacin (20μM) or sulfinpyrazone (ImM) reduced 14C-5HT release in response to collagen from 45-65% to 5-8%, completely blocked MDA formation and reduced the amount of PGF in the supernate to 33-44% of control values. These inhirbitors reduced release caused by thrombin (5U/ml) from 90-95% to 85-88% and, although they completely blocked MDA formation, did not decrease PGF. PGF was not made available by aggregation of platelets with ADP.(Release does not occur with rabbit platelets exposed to ADP). Thus, the appearance of PGF does not depend on primary aggregation but is associated with the release reaction and is independent of the endoperoxide pathway.
Gamma Camera Detection of Experimental Pulmonary Emboli After Peripheral Injection of Indium-111 Labeled Platelets
Authors: McIlmoyle G. , David H.H. , Welch M.J. , Primeau J.L. , Siegel B.A. , Sherman L.A.
Published: May 2019

A simple, noninvasive method for the direct visualization of pulmonary emboli would be of considerable value for the diagnosis of this common disorder, especially in patients who have underlying parenchymal lung disease or are too ill to undergo pulmonary angiography. For this purpose, we have investigated the accumulation of In-111 platelets in acute pulmonary emboli. Radiolabeled venous thrombi were produced in 6 dogs by the injection of human thrombin and Tc-99m sulfur colloid into occluded segments of both jugular veins. One hour later, the thrombi were released and gamma camera images demonstrating the position of the Tc-99m labeled pulmonary emboli were obtained. Autologous platelets labeled with In-111 oxine were then injected peripherally and sequential images were obtained for one hour. The dogs were then sacrificed, and the emboli and tissue samples were removed and assayed for activity. Sixteen pulmonary emboli containing Tc-99m sulfur colloid were seen by imaging and 14 of these were detected with In-111 platelets; 5 emboli were visualized immediately and uptake in the remaining 9 appeared on later images. In all animals, at least one embolus was detected by imaging. Mean embolus weight was 538 mg, mean In-111 uptake was 1.09% dose/g embolus, and mean embolus/blood ratio was 16.1. In 2 animals, images showed additional foci of increased In-111 uptake, distal to an embolus containing Tc-99m; this uptake may reflect thrombus propagation. Our results demonstrate that acute pulmonary emboli in dogs can be readily detected by imaging with In-111 labeled platelets, injected after embolization.
Macromolecules Released from Platelet Storage Organelles
Authors: Pepper D.S. , Scotland S.E.
Published: May 2019

Much attention has been paid to the low molecular weight substances released from platelets since the release reaction was first discovered. The release of macromolecules on the other hand has received much less attention, and of the published work on platelet macromolecules most has been devoted to the known plasma protein eauivalents which are either synthesized de-novo or adsorbed by the platelet prior to their subsequent release. Whereas it is fairly easy to show that the physical and/or immunological properties of these molecules (notably albumin, fibrinogen and IgG) are slightly but reproducibly different from those of plasma, it is very difficult (if not impossible) to prove that they are truly synthesized within the platelet as such, rather than undergoing modulation during the release reaction or isolation procedure. Whilst these “plasma equivalent” proteins are of academic interest, they have less practical application than those which are “platelet specific” i.e. are not normally found in native, platelet free plasma. Unfortunately, there are several technical problems in the study of such proteins, and we have chosen to study them by a combination of techniques, namely biological activity, immunological activity and physico-chemical properties. Each approach has some drawbacks, but in combination, much progress is possible and we can now identify at least four antigens, four biological activities, and two physically distinct proteins, which are not necessarily related. With the recent interest in the biological activity of released macromolecules, it is now desirable that the various groups exchange samples and antisera in order to identify each antigen and biological activity.
Primary Structure of Human Platelet Factor 4.
Authors: Morgan F.J. , Begg G.S. , Chesterman C.N.
Published: May 2019

The amino acid sequence of human platelet factor 4 (PF4) has been studied. PF4 is a platelet specific protein with antiheparin activity, released from platelets as a proteoglycan complex, whose measurement may provide an important index of platelet activation both in vivo and in vitro. These studies were undertaken to characterize fully the PF4 molecule. PF4 is a stable tetramer, composed of identical subunits, each with a molecular weight based on the sequence studies of approx. 7,770. Each PF4 subunit contains 69 amino acids, including 4 half-cystine (# 10, 12, 36, 37), one tyrosine (# 59), 3 arginine and 8 lysine, but no methionine, phenylalanine or tryptophan residues. The basic residues are predominantly in the C-terminal region. The tryptic peptides were aligned after studies which included tryptic digestion of citraconylated RCM-PF4, and automated Edman degradation of RCM-PF4 and citraconylated tryptic peptides. No glycopeptides were detected. This structural information should enable clear distinction to be made between PF4 and other platelet proteins such as β thromboglobulin. The provisional amino acid sequence of each subunit is:

Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Cys-Pro-Thr-Ala-Gln-Ile-Leu-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Pro-Leu-Asp-Leu-Gln-Ala-Tyr-Leu-Lys-Ile-Lys(Lys, Lys, Ser, Glx, Leu, Leu)
Fibrinogen/Fibrin Interaction with Platelets
Authors: Michaeli D. , Orloff K.
Published: May 2019

There is considerable evidence that fibrinogen is involved in ADP-induced platelet aggregation. On the basis of fluorescent probe experiments, it was concluded that fibrinogen increases the exposure of hydrophobic regions on the platelet membrane. A proposal was made that fibrinogen enhancement of ADP-induced aggregation is mediated via a thrombin-fibrinogen interaction leading to the formation of fibrin bridges between platelets. Evidence militating against this mechanism will be reviewed.

Platelets are capable of interacting with nascent fibrin, which in turn induces the aggregation and release reactions. Platelets can similarly react with fully polymerized fibrin. Fragment D inhibited these interactions, probably due to its binding to fibrin, and thereby blocking sites on fibrin that are involved in its interaction with platelets.
Complications of Extracorporeal Perfusion
Authors: Rodvien Robert
Published: May 2019

Presently, extracorporeal circulation (ECC)is being used for cardiopulmonary bypass, and to support individuals with liver, renal or lung failure. The recognized clinical complications of ECC are primarily bleeding, thrombosis and microvascular occlusive disease. These complications are always modified by the patient’s underlying illnesses and by the technique of ECC (the quality of the foreign surface, the perfusion, and the surgical intervention required). The abnormal surface to which blood is exposed may be the actual polymer of the device, the gas bubbles of the oxygenator or the altered endothelium induced by the disease. Because foreign surfaces are thrombogenic, all techniques of ECC require heparin to inactivate the coagulation proteins and prevent thrombosis. Despite heparin, platelets can be retained by the surface transiently or permanently, and release of pharmacologically active materials from platelets and leucocytes is expected. Other surface-activated portions of the blood have not received as much attention, but they may produce significant morbidity. These other elements include the kinin, complement, and fibrinolytic systems. The immediate and most overt complications of bleeding and thrombosis as well as specific long-term complications of ECC such as neurologic dysfunction will be discussed in the context of how ECC produces alterations in blood cells, proteins and lipids.