bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Primary Structure of Human Plasminogen and Plasmin, Cyanogen Bromide Fragments
Authors: Wiman B. , Wallén P.
Published: June 2019

Human plasminogen is a single chain molecule with a molecular weight of 93,000. This protein contains 8 methionine residues per mole. Cleavage with cyanogen bromide and subsequent reduction and S-carboxymethylation resulted in about 15 fragments ranging in size from 11 residues to about 310 residues. Most of these fragments have been purified by combinations of gel filtration and ion exchange chromatography. Characterization by molecular weight determination, amino acid composition, carbohydrate analysis and direct NH2-terminal amino acid sequence determination were thereafter performed. Some of the fragments were found to be the result of incompletely cleaved methionyl bonds which in a very convenient way allowed us to elucidate almost the complete organization of the cyanogen bromide fragments. — Plasmin is a two-chain molecule with a molecular weight of 85,000 formed from plasminogen by activation with urokinase involving proteolytic cleavage in two steps. The first step is release of the NH2-terminal 68 residues and the second step cleavage of an arginyl-valyl bond at about residue number 600 during formation of the two-chain structure. The cyanogen bromide fragment which contains that arginyl-valyl bond and thus forms the linkage between the heavy (A) and light (B) chains, consists of 117 amino acid residues. Its primary structure is almost completely determined. The NH2-terminal 93 residues form the carboxyterminal part of the heavy (A) chain and the carboxyterminal 24 residues form the NH2-terminal part of light chain. Furthermore this fragment and another fragment, which forms the major part of the light (B) chain, show a microheterogeneity which is very similar to that found in the intact plasminogen molecule. Neither of these two fragments seems to contain any sialic acid, which instead seems to be concentrated together with most of the hexoses to another heavy (A) chain fragment, which is very difficult to purify.
Positive Chronotropic Effect of Dialysable Peptides Derived from Plasmin Digestion of Bovine Fibrinogen Preparations. Role of Cyclic AMP
Authors: Buczko W. , Franco R. , Bianchetti G. , Donati M. B. , Gaetano G. de
Published: June 2019

Dialysable peptides (< 5,000 M. W.) derived from plasmin digestion of bovine fibrinogen preparations (Kabi) exerted a marked positive chronotropic effect on isolated rat atria. This effect reached a peak within two to four minutes, lasted as a plateau during four to six minutes and then slowly disappeared. It was abolished by glycolytic inhibitors such as NaF and 2-deoxy-D-glucose, not by inhibitors of oxidative phosphorylation such as 2,4-dinitrophenol. Ouabain, histamine and propranolol had no effect on the digests-induced chronotropic effect. Theophylline, an inhibitor of phosphodiesterases, showed an additive effect, whereas imidazole, an activator of phosphodiesterases, was inhibitory. Both cyclic AMP levels and phosphorylase activity of isolated rat atria were increased by the digests. The maximum increase was observed 30 and 90 seconds respectively, after the addition of the digests to the isolated atria. Theophylline exerted an additive effect and imidazole an inhibitory effect on digests-induced increase of cyclic AMP levels and phosphorylase activity. In our test system, digests behaved similarly to glucagon. It is concluded that cyclic AMP plays an important role in mediating the described positive chronotropic effect of fibrinogen-derived peptides. Such a role of cyclic AMP could also be relevant for other biological activities exerted by these peptides (e.g. the inhibition of ADP-induced platelet aggregation and the increase of vascular permeability and of the chemotactic activity of leucocytes).

(Supported by Grant N. 73.00400.04 of the Italian Research Council (C. N. R.).)
Flaujeac Trait: Deficiency of Human HMW Kininogen
Authors: Lacombe M. J. , Miller D. R. , Wuepper K. D.
Published: June 2019

The propositus, a 50 year-old woman of French Caucasian extraction, was the product of a consanguineous marriage. Her plasma, obtained at the time of a surgical procedure, exhibited prolonged whole blood clotting time and prolonged activated PTT (618 sec); the one-stage prothrombin time was normal. Her plasma contained normal amounts of coagulation factors XII, XI, IX, VIII, VII, X, V, II, I and Fletcher factor. Full correction was obtained upon mixing with normal plasma, excluding a circulating anticoagulant. The defect of the propositus, surname Flaujeac, was designated Flaujeac Trait. Three of the patient’s children had reduced amounts of the factor (23%, 27%, 30%) while a fourth child was at the lower limit of normal (55%).

Flaujeac trait plasma was also found to have prolonged euglobulin lysis (194 min), failure of kaolin-activated kinin formation or generation of the permeability globulin PF/dil. Each of these deficits was corrected by addition of a factor separable from Hageman factor or Fletcher factor (prekallikrein). The Flaujeac factor was identified as a heat stable, α-globulin which eluted from Sephadex G-200 corresponding to a MW of 170,000. The factor corresponded exactly with fractions containing HMW kininogen. The half-life in plasma following transfusion was 6.5 days. Kininogen antigen was markedly reduced in Flaujeac plasma, only some partially cross-reacting material remained. Antibody to kininogen depleted the factor from normal plasma.

The intrinsic coagulation, fibrinolytic and kinin-forming pathways and the formation of PF/dil are dependent upon plasma HMW kininogen.
Interactions between Factors XII and XI and Activating Agents in Purified Systems
Authors: Binder B. , Krug G. , Vukovich Th.
Published: June 2019

To obtain a better understanding of the activating mechanism of F XI we studied the interaction of highly purified F XII and F XI with activating agents (kaolin, cephalin) F XII as well as F XI were purified from ACD-plasma (ionic exchange procedures, gel fractionation). Both coagulation factors were obtained with a specific activity per mg protein of approximately 4000 times the value of the original plasma, showing a single band in polyacrylamide gel electrophoresis. The preparations contained less than 1 per cent of their activated forms.

F XII and F XI as well as the mixture of both were exposed to kaolin, cephalin, or a kaolin-cephalin mixture, respectively. F XII was adsorbed to about 90 per cent by kaolin alone and the adsorbed fraction was activated completely within minutes; F XI on the other hand was neither adsorbed nor activated by kaolin. The kaolin-activated F XII effected practically no activation of F XI. By cephalin F XII was activated only within hours, but the so activated F XII produced activation of F XI corresponding to F XIIa available in a ratio F XIIa/F XI a of 10/1. A mixture of kaolin and cephalin activated F XII within minutes; ca 20 to 30 per cent of the resulting F XIIa activity became not adsorbed to kaolin and this F XII a activates F XI in the same ratio as in the case of cephalin-activated F XII. Gel fractionation of F XII and F XI together with cephalin revealed that only F XIIa is bound to the phospholipid; no solid complex formation was found between F XI and cephalin or between F XI and F XII.

(Supp. by a Grant from the Eisner-Foundation.)
Heparin Neutralizing Activity in the Diagnosis of Acute Myocardial Infarction
Authors: O’Brien J. R. , Etherington M. D. , Jamieson S. , Sussex J.
Published: June 2019

We have previously demonstrated that, relative to controls, patients long after myocardial infarction and patients with atherosclerosis have highly significantly shorter heparin thrombin clotting times (HTCT) using platelet poor plasma; but there was considerable overlap between the two groups.

We have now studied 89 patients admitted with acute chest pain. In 54 of these a firm diagnosis of acute myocardial infarction (ac-MI) was made and the HTCT was very short (mean 12.8 sees) and in 48 it was less than 16 sees. In 34 patients, ac-MI was excluded and the diagnosis was usually “angina”; the HTCT was much longer (mean 25.1 sees) and in 32 it was over 16 sees. Thus there was almost no overlap between these two groups. It is suggested that this test should be adopted as a quick and reliable further test to establish a diagnosis of ac-MI (providing other reasons for very short HTCTs can be excluded, e.g. D. I. C., and provinding the patient’s thrombin clotting time is normal).

This HTCT measures non-specific heparin neutralizing activity; nevertheless the evidence suggests that it is measuring platelet factor 4 liberated from damaged or “activated” platelets into the plasma. These findings underline the probable important contribution of platelets in ac-MI.
Evidence for Separate Mechanisms of Induction of the Platelet Release Reaction by Collagen and Adrenaline
Authors: Tangen O. , Bygdeman S.
Published: June 2019

The effect of some selected inhibitors of platelet release reaction and blood clotting on collagen- and adrenaline-induced human platelet aggregation was investigated by means of the turbidimetric method according to Born. Acetylsalicylic acid (ASA) inhibited both collagen- and adrenaline-induced platelet aggregation in citrated platelet rich plasma (PRP). Addition of sufficient amounts of Ca++ to give concentrations similar to those in native blood suppressed the inhibition by small doses of ASA (5–10 μg/ml) on collagen-induced aggregation and the second phase of adrenaline-induced aggregation. Higher concentrations of ASA (13–30 μg/ml) could partly overcome this effect of Ca++. Heparin, which had no effect on primary adenosine diphosphate (ADP)-induced aggregation, inhibited platelet aggregation induced by collagen. In contrast, both the first and second phase of adrenaline-induced aggregation was markedly potentiated by heparin. Dextran sulphate had effects basically similar to heparin, Nicotinic acid inhibited collagen-induced aggregation, but had no effect on the second phase of adrenaline-induced aggregation. These results indicate that the platelet release reaction induced by collagen and adrenaline is mediated via separate receptors or reaction pathways.
Study of Albinism in Relation to Hermanský-Pudlák Syndrome
Authors: Hermanský F. , Cieslar P. , Matousová O. , Smetana K.
Published: June 2019

Up to now 24 patients with oculocutaneous albinism have been investigated in this study to find out the frequency of characteristic signs of HPS. Seven of them suffered from mild bleeding tendency. Platelet defects of storage pool disorder type have been revealed at a varying degree in all these cases. (Aggregation abnormalities, decrease of total platelet ADP contents and of its release, lower serotonin uptake with the lack of very dense bodies in ultrathin sections). This thrombocytophathy has been regularly associated with the storage of pigment typical for HPS in their bone marrow. In 7 of 17 remaining albinos with normal platelet functions some increase in the accumulation of non-specific cytochemically different lipopigments was noted.

Our results demonstrate a rather frequent occurence of HPS in albinos and suggest a mutation of one common gene or of closely linked genes as a cause of complete HPS.
The Effects of Natural Oestrogens on Blood Clotting
Authors: Poller L. , Thomson Jean M.
Published: June 2019

A double-blind cross-over trial on natural conjugated oestrogens (Premarin) has been conducted in women volunteers. Base-line studies were performed on blood clotting and platelet function and these were repeated at 3 month intervals during therapy. Laboratory results and possible thrombogenic implications are discussed.
Amine Inhibition of Fibrin Cross-Linking
Authors: Dubiel M. K. , Scully M. F. , Lane D. A. , Kakkar V. V.
Published: June 2019

Various amines have been shown to possess an inhibitory effect on the enzymatic cross-linking of fibrin in vitro, as measured by fibrin isolubility in monochloroacetic acid (Lorand, L. et al., Biochemistry, 7, 1214, 1968). The amines were found to act as pseudo-donor substrates for the enzyme Factor XIIIa.

We have measured γ dimer and α polymer formation in vitro, using 125I-fibrinogen as a tracer. A solution of 0.4% fibrinogen clotted with thrombin at pH 7.5 produced 50% of the γ dimers within 2 seconds and 65% of the oc polymers in approximately 15 minutes. In the presence of 5 mM isoniazid, glycine methyl ester, histamine and putrescine, 60-70% inhibition of α polymer formation was observed at 15 minutes, as compared with the control incubation mixture. There was minimal inhibition of γ dimer formation. Tyramine and histidine dihydrochloride had no inhibitory effect upon either γ dimer or α polymer formation.

These results provide further evidence that a carboxylate anion in the vicinity of the amino group reduces inhibition of Factor XIII a and suggests that inhibition as measured by acid insolubility is also influenced by α polymer formation.
Fibrin-Blood Platelet Interaction in a Contracting Clot
Authors: Cohen I. , Gabbay J. , Glaser T. , Oplatka A.
Published: June 2019

The interaction of the fibrous protein fibrin and blood platelets was studied by measuring tension development in clots containing different ratios of these two components. Maximal isometric tension (MT) attained a saturating value when plotted as a function of the concentration of either component at a constant concentration of the other. The value of MT increases linearly with the minimal platelet concentration (Ps) required in order to reach saturation at various different fibrin concentrations. A linear relationship also holds in a plot of Ps versus the logarithm of the corresponding fibrin concentration. An attempt was made to calculate the isometric tension developed by a platelet.

When the cylindrical clots contracted isotonically, the velocity normalized with respect to the momentary length was found to be constant. This velocity decreased in an hyperbolic manner with increasing fibrin concentration at a constant concentration of blood platelets. The results are interpreted in terms of a three-dimensional network in which the blood platelets serve as contractile cross-bridges connecting fibrin fibers.
Effects of pH on Arachidonic Acid Induced Platelet Aggregation
Authors: Gerrard J. M. , White J. G. , Krivit W.
Published: June 2019

Arachidonic Acid (AA) produces platelet aggregation through conversion to Prostaglandin (PG) G a and PGH2. The present study evaluated the effect of pH on AA induced aggregation and production of PGG2 and PGH2. AA was added to 1 ml containing 2 × 108 washed platelets. The pH of the reaction was adjusted from pH 6.5 to pH 9.0 using C02 or NaHC03. Aggregation was monitored by recording nephelometry.

Production of PGG2 and PGH2 was evaluated at 30 seconds after addition of AA by SnCl2 conversion of the endoperoxides to PGF2Ct and subsequent radioimmunoassay. Production of 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), 12L-hyd,roxy-5,8,10-heptadecatrienoic acid (HHT), and 8-(l-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (PHD), three metabolites of AA, was monitored using 1-14C AA and thin layer radiochromatography. The optimum pH for AA induced platelet aggregation and for production of PGG2 and PGH2 was 8.0. Maximal production of PHD similarly occurred at pH 8.0. However, production of HHT was maximal at pH 9.0 and production of HETE was maximal at pH 6.5. The results show that the platelet prostaglandin synthetase has a pH optimum of 8.0 similar to the sheep vesicular gland synthetase and suggests an enzymatic basis for the finding that the pH optimum for collagen induced aggregation of washed platelets is 8.0. The results also suggest that production of PHD, but not HHT or HETE, is closely linked both to platelet production of PGG2 and PGH2 and the degree of platelet aggregation.
The Isolation of Prothrombin Fragments from Plasma Clotted with Russell Viper Venom (RVV)
Authors: Aronson D. L. , Stevan L. , Ball A. P. , Franza R. B. , Young A. M.
Published: June 2019

Plasma clotted with RVV (1 mg/L) for 90 minutes loses 60% of the measurable prothrombin. Batch adsorption of the serum with DEAE-cellulose followed by hydroxyapatite chromatography and gel filtration on Sephadex G-200 yields 4 mg/L of Fragment F1.2, the prothrombin NH2-terminal activation peptide of mass = 35,000 daltons. Identification is based on migration on SDS acrylamide electrophoresis and the ability of small amounts of thrombin to cleave F1.2 into F1 and F2.

The presence of Fragment F1.2 after clotting plasma in this manner demonstrates that the average amount of thrombin present during clotting was less than 1 u/ml. This fact in combination with 60% prothrombin consumption indicates that the thrombin formed is inactivated in less than one minute.

F1.2 is soluble in 0.9 M perchloric acid, as are F1 and F2. This property may simplify the purification process and allow quantitation of F1.2 release during clotting.
Prothrombin Activation
Authors: Greenberg J. , Goldstein R.
Published: June 2019

Prothrombin DFP treated (II) when activated by Xa in the presence of 10-2 M DFP, generated 0.1 Iowa unit of active Thrombin (IIa) per one mg of II (IIa is 10-3 of the weight of Xa present); 26% of the original II was converted to inactive IIa- Reduction of the activation mixture produced chains of 42-57,000 daltons. During the first 90 sec of the lag phase of that reaction, peptides and/or amino acids were released. During the first hour of II activation by Xa in the presence of 10-5 M DFP (weights of active IIa ÷ f Xa = 0.9) transient intermediates of M. W. of 65-74,000 were observed. These findings indicate the following Xa activation of II pathway : II (one chain) → intermediate (s) (two chains) + peptides or amino acids → fragment(s) + thrombin(s) (two chains). The proposed pathway is consistent with earlier reports of production of fragments of 37,000 and 45,000 daltons, the multiple forms of II-, the rate and the yield of II conversion to IIa via its physiological phospholipoprotein complex and also of the kinetic rate requirement that II be attached to that complex, through its N-terminal portion, until the last step of IIa production. During the first ten min of II activation by Xa, in absence of DFP (IIa ÷ Xa = 2), the production of IIa was found to proceed partially via above Xa activation pathway. As the amounts of IIa increased, the IIa pathway (via Int. 1, 60,000 daltons) exceeded that of Xa pathway.

(Supported by N. I. H. Grant # HL-14657.)
A Screening Method for Deep Venous Thrombosis
Authors: Jaffrin M. Y. , Michaux B. , Quarante J. J.
Published: June 2019

This paper presents some preliminary results on a non invasive technique for the detection of deep venous thrombosis. This method is simple, completely atraumatic and capable of detecting silent thrombosis which eludes clinical examination. It also integrates the contribution of all veins in the limb in a single measurement. The principle of the method consists in interrupting the venous return in the thigh by a cuff inflated to pressures ranging from 30 to 50 mmHg. The subsequent increase in distal venous volume is monitored on the calf by a four-electrode impedance plethysmograph or by a mercury-in-silastic straingage plethysmograph (Parks) IPG 100 of Codman or Diacardyne of Ieram). The trace representing the limb volume or perimeter reaches a plateau after a period of about a minute when the rising venous pressure reaches the pressure in the thigh cuff, and the venous return is restored. Then the thigh cuff is rapidly deflated and the rate at which the limb volume returns to the preocclusion level is recorded. The signal decays approximately exponentially with time with a time constant equal to RC where C is the venous compliance and R the venous flow resistance downstream of the cuff. The time constant ranges from. 7 to 2 sec for healthy veins. If a large vein is thrombosed the flow resistance is higher and the time constant exceeds 2.5 sec. The method has being tested on about 50 patients of the Centre Hospitalier of Compiégne and compares favorably with clinical methods or with Doppler ultrasound if the leg and cuff are properly positioned. The average time constant was found to be equal to 1.75 sec for patients known to be free of thrombosis and to 4.0 sec for patients with confirmed venous thrombosis. Present work is directed towards improving the instrumentation. The determination of the time constant is made easier by filtering out arterial pulsations superposed on the signal. By using an automatic inflation with preset inflation pressure and duration accurate and reproducible inflation sequences of tho cuff are possible.

This may lead to additional diagnostic criteria based on the rate of signal rise.
Venous, Arterial and Endocardio Multiple Thrombosis with Consumption Coagulapathy and Cryofibrinogen due to Ovarian Carcinoma Temporary Improvement with Heparin
Authors: Favre-Gilly J. , Michel D. , Tommasi M.
Published: June 2019

A 54 years old woman successively developed thrombophlebitis of lower limbs, cerebral thrombosis with right hemiplegia, coronary thrombosis and arterial thrombosis of upper and lower limbs. Haematuria and large bruises led to find thrombopenia and lower levels of fibrinogen, prothrombin, factors V and VIII, with cryofibrinogen. Heparin stopped haemorrages and defibrination and improved thrombosis during 2 weeks, but the patient died of recurrent thrombosis extended to upper vena cava. Post mortem examination allowed to find many thrombi on endocardium and in large and small vessles, both arterial and venous. There were many infarcts in brain, myocardium, splen, and left kidney. Ovaries were both carcinomatous.
Ultrastructural Changes in Human Platelet During Fatty Acid Salt Aggregation
Authors: Prost-Dvojakovic R. J. , Men R. Le , Soria J. , Boisseau M.
Published: June 2019

We studied ultrastructural changes in platelet aggregation induced by fatty acid (FFA) salts. The platelets were used either directly in their original plasma or separated from plasma proteins by washing or filtration on 2 B Sepharose gel and suspended in a buffer solution with or without albumin.

After fixation for electron microscopy the aggregating FF A salts appear as micellar structures or spherules of large size ressembling liposomes.

During the first seconds of contact between the platelets and the FFA salts we observed the spreading of platelets on the surface of the structures formed, and the particles smaller than 1 μ were phagocytized. After a few minutes platelet release occurred followed by total degranulation of platelets during the formation of aggregates. After eight minutes the aggregates still contain FFA salts structures.

These results confirm our earlier views that the mechanism of FFA salt induced aggregation differs from that of aggregation by other agents, and could be due to the micellar structure.
Indications and Results of Fibrinolytic Treatment with Streptokinase in Chronic Arterial Stenoses and Occlusions; a Multicentre Trial of 708 Patients
Authors: Schmutzler R.
Published: June 2019

In 7 German medical centres fibrinolytic therapy with streptokinase was used in 708 patients with subacute or chronic arterial stenoses or occlusions. Detailed opinion would be reached if one regards stenoses separately from occlusions, if one takes special localisation of blood vessel changes as well as the period of claudication into consideration. After the indications had been narrowed down in time, the limits for the ability to lyse were set: in the case of femoropopliteal vessels to 8 weeks, occlusions of pelvic arteries to 1 year, and for stenoses in this region to 2 years. No time limits were made in the terminal aorta. When taking these limits into consideration, it was clearly shown that the success rate increased in the case of vascular stenoses from 59 to 66%, and in occlusions from 22 to 39%. The mortality rate was 1.3% compared to 1.0% of patients untergoing surgery. Fatal cerebral bleeding occured in 0.7%.
Electron Microscopic Studies of the Molecular Shape of Fibrinogen Molecule
Authors: Tranqui L. , Marder V.J. , Suscillon M. , Budzynski A. Z. , Hudry G.
Published: June 2019

Electron microscopic studies of fibrinogen have demonstrated many variations in shape and size, with a corresponding number of opinions regarding its transformation to fibrin. To this end, we have subjected fibrinogen to a variety of stimuli and have studied the changes that follow, by negative staining electron microscopy. At relatively physiologic conditions to pH 7 and ionic strength 0.15, the molecule assumes a roughly spherical shape. Denaturation with SDS results in an elongated molecule of approximately 900 Å length. Noncoagulable products obtained after plasmin exposure appear also as elongated filaments, the largest of which has a molecular weight about 20% less than the intact molecule and a length of about 600 Å. Thrombin-induced fibrin formation shows a series of molecular alterations compatible with a structural reorganization of these filaments. After partial degradation of fibrinogen with proteolytic enzymes (plasmin, bacterial proteases), thrombin treatment results in fibrin with the same major periodicity of 23 nm, but different minor striations, compatible with the loss of different portions of the molecule.
Experimental Modification of the Platelet Release Reaction Induced by Collagen
Authors: Gordon J. L. , Maclntyre D. E. , Drummond A. H.
Published: June 2019

Collagen-induced release of platelet constituents can be divided into two kinetically-distinct phases, only one of which is associated with platelet aggregation, and the aggregation-independent release is less susceptible to inhibition by pharmacological agents (Drummond and Gordon, 1974). Minor variations in experimental conditions alter the release reaction profile. Collagen was incubated with platelet rich plasma (PRP) for one minute at 37°, under conditions in which no aggregation occurred ( + 3nM EDTA or in absence of stirring). The reaction was ‘terminated’ by addition of ice-cold EDTA-saline and samples were then centrifuged (14,800 g) under the conditions described in the table.

No significant release was detected under any of the above conditions if saline were substituted for collagen suspension.

These results indicate that the storage temperature and time after centrifugation are as important as the centrifugation conditions themselves, and suggest reasons for discrepancies reported in previous studies of collagen induced release kinetics.

Drummond, A. H. and Gordon, J. L. (1974). Brit. J. Pharmacol. 52, 130 P.
Leucocytes and Platelets in Isolated Lung Perfusion
Authors: Zandwijk N. van , Lenssen Th. F. J. , Prakke Elisabeth M. , Meer J. van der , Groen A. S. , Wagenvoort G. A.
Published: June 2019

Removal of leucocytes and platelets from homologous blood prevents early functional and morphologic deterioration of an isolated rabbit lung preparation. Lungs perfused with whole blood have a marked ability for granulocyte sequestration. Control experiments in which the perfusion system was not connected with the lung revealed an increase of plasma serotonin and histamine levels due to platelet injury and/or aggregation generated by the perfusion circuit. When an isolated lung was connected with the circuit, plasma serotonin was largely metabolized. However, the metabolic uptake of serotonin by lungs perfused with whole blood decreased after 30 min while pulmonary vascular resistence (PVR) increased. In contrast, perfusion experiments with blood from which almost all leucocytes had been removed, were characterized by a less pronounced rise of plasma serotonin level and no or little change in PVR.

It is concluded that leucocytes particularly granulocytes most likely traumatized by the extracorporeal procedure, have a detrimental effect on the integrity of perfused isolated lungs. Damage of endothelium, which has been recognized as a site of serotonin metabolism, could then be an early symptom. The results of our experiments in which the perfused isolated lung preparation has been used as a model for pulmonary dysfunction after extracorporeal circulation and massive transfusion warrants more attention to be paid to the role granulocytes and platelets play in the genesis of respiratory distress after such procedures.
Platelet Aggregation in whole Blood
Authors: Sivertsen Ulla
Published: June 2019

A semi-micromethod for testing the aggregability of platelets in whole blood is described. Aggregation is induced with ADP at 37° C in 0,1 ml samples of citrated blood. At 30 seconds intervals aggregation is stopped and the number of nonaggregated platelets are determined.

The main results as found in “normal” human donors (having received no drugs) are:

1) Platelet aggregation in whole blood is less dependent on the pH, the platelet number, the time elapse from blood sampling to aggregation test as compared with platelet aggregation in plasma (a.m. Born).

2) Disaggregation is only observed to a minor degree (after 180 seconds the maximal disaggregation amounts to 30% of the aggregated platelets).

3) Secondary aggregation is negligible.

The technique is simple and can be used in the clinic without special equipments. The results are discussed.
Light Scattering and Platelet Aggregation
Authors: Stoltz J. F. , Larcan A. , Batoz J. F. , Streif F.
Published: June 2019

After a short recall of the theories concerning nephelometry and light scattering, the authors develop their experimental study, which is divided into three parts :

- platelets absorption i.e. wave lengths

- light variation curves transmitted or scattered according to platelet concentration

- aggregation by nephelometric method and by light scattering.

These experiments allow the authors to conclude that platelets do not present a specific absorption; that the variations of the light transmitted or scattered is exponential in function of the platelet concentration and that the aggregation test affords essentially a measurement of the decrease of the number of free platelets in the medium.

Besides, they observe that the measures of aggregation kinetics, or the problem of platelet shape change are not specific and should be investigated with the help of other methods.

This work was supported by D. R. M. E. grant (Biological Section).
Diagnostic Information by Haemorrhagometry
Authors: Sutor A. H.
Published: June 2019

A new vivo method to measure bleeding time, bleeding intensity and blood loss from a standardized small skin wound was developed by Sutor, Bowie, and Owen at the Mayo Clinic. At room temperature (24° C) bleeding time is prolonged in patients with quantitative and qualitative platelet deficiencies. Patients with Morbus Willebrand show a prolonged bleeding time and an abnormally high bleeding intensity. Patients with reduced capillary resistency (M. Davis and allergic diathesis) have increased bleeding intensity but normal bleeding time. Aspirin and Dipropyl-Acetate increase the blood loss, prednisone reduces the bleeding intensity. When haemorrhagometry is performed at 17° C (cold tolerance test) bleeding time is pathologically increased in haemophiliacs and in most carriers of haemophilia. If an already closed wound is heated to 43.5° C it starts bleeding again in control subjects, the average heat lysis time is 1.03 min (SD : 0.16 min). In patients with thrombocytic haemorrhagic tendencies the heat lysis time is shortened; a connection between prolonged heat lysis time and hypercoagulaemia has been shown in some cases.
Variations in Quantitative Ristocetin Platelet Aggregation (RPA) with Time after Platelet Rich Plasma (PRP) Preparation: Association with Alteration in pH
Authors: Coller B. S. , Gralnick H. R.
Published: June 2019

Decreased quantitative RPA of PRP is used to distinguish patients with von Wille-brand’s disease from normals and hemophiliacs. We observed that RPA (1.8 mg/ml final concentration) of PRP from 9 normals varied markedly with time after PRP preparation. In 6/9 the initial RPA was greatest and decreased progressively with time. In 3/9 RPA increased over the first half hour and then decreased progressively. The decrease from peak values was 37 ±12% (mean±1 SD) at 1 hr, 69 ±20% at 2 hr and 70±10% at 2.5 hr.

Several data suggest a role for pH in this phenomenon: (1) pH of the PRP’s increased with time and the decreases in RPA paralleled the increases in pH (2) maintaining the PRP under a 5% CO2-95% O2 atmosphere prevented the rise in pH and lessened the decrease in RPA over time (3) rapidly increasing the pH to 8.3 by bubbling nitrogen through the PRP or titrating with base decreased RPA (81% and 55%) (4) immediate back titration restored the RPA.

We conclude that quantitative RPA changes with time after PRP preparation and this is probably in part a function of pH. Since pH change with time is variable for different PRP preparations, choosing an arbitrary time limit after PRP preparation for performing studies is an inadequate control. Quantitative RPA should be pH controlled.
Interaction of Ristocetin (R), yon Willebrand Factor (vWF), Factor VIII Clotting Activity (VIIIc) and Platelets
Authors: Zucker M. B. , McPherson J. , Kim S.-J. , Macdonald V. E. , Taylor D. , Johnson A. J. , Barlow G. H.
Published: June 2019

Formalin-fixed platelets were resuspended in a 1 in 3 dilution of plasma and aggregated with 1.5 mg 3H-R per ml. The supernatant contained over 90% of the R, about 25% of the vWF (assessed as R co-factor or by radioimmunoassay of VIIIag) and 50% of the VIIIc. The aggregates contained 6-9% 3H-R and only 1-2% of trapped 14C-albumin. They dispersed in buffer in the absence of R, and about 35% of the initial vWF but only 16% of the initial VIIIc was recovered in the resuspending buffer. Thus the ratio of vWF to VIIIc activity was < 1 in the initial supernatant and > 1 in the buffer in which the aggregates had been dispersed. Platelets in PRP behaved similarly but bound only a small percentage of plasma vWF during aggregation, and less R than formalin-fixed platelets. The latter bound R in the absence of plasma. Bernard-Soulier platelets did not aggregate; they bound R but not vWF. When 3H-R was added to fresh normal plasma and passed through a Sepharose-4B column, R failed to appear in the void volume (Vo) but emerged earlier than when chromatographed without plasma. vWF and VIII emerged together in Vo when plasma alone was used, but VIIIc peaked later than vWF in the presence of R. In summary, (1) R separates VIIIc from vWF, (2) platelets are passively involved, (3) R binds reversibly to normal as well as to Bernard-Soulier platelets, (4) in the presence of R, vWF binds in part reversibly to normal but not to Bernard-Soulier platelets. Thus separation of vWF and VIIIc by R may produce altered vWF which alone cannot bind R or aggregate platelets, but can bind to and hence aggregate platelets in the presence of R.
Inhibition of Bovine Factor VIII and Ristocetin Plus Human Factor VIII Induced Platelet Agglutination by Small Peptides
Authors: Macfarlane D. E.
Published: June 2019

The antibiotic ristocetin induces platelet agglutination in the presence of human factor VIII. A similar agglutination is induced by bovine factor VIII without ristocetin. This antibiotic binds C-terminal D-alanine peptides including the nucleotide-N-acetyl-muramyl-peptide precursor of bacterial cell walls. The effect of several peptides on the agglutination of formalin fixed washed human platelets was investigated. At 1.25 mM acetyl-glycyl-D-alanine (AGDA) and diacetyl-L-lysine-D-alanine-D-alanine (ALAA) increased the amount of ristocetin required for agglutination in the presence of purified human factor VIII; both these peptides bind to ristocetin (Bichochem. J. 124, 845). Glycyl-L-alanine (GLA) glycyl-D-alanine (GDA), acetyl-glycyl-L-alanine (AGLA) were inactive. AGLA (0.25 mM) and AGDA (1.25 mM) inhibited purified bovine factor VIII induced agglutination, ALAA, GLA and GDA (3.2 mM) were inactive.

These results suggest that ristocetin acts in part by binding to peptides on the platelet surface, and that bovine VIII combines to the same site but has a different specificity. Suitable small peptides may be able to induce a von Willebrand like state for therapeutic purposes.

(Dr. Kirby, Temple University provided the purified factor VIII’s and Prof. Perkins, Liverpool University provided ALAA; supported by N. I. H. HL 14217.)
Estimation and Characterization of Soluble Fibrin monomer Complexes (SFMC) During Endotoxin Induced Disseminated Intravascular Coagulation (DIC)
Authors: Hafter R. , Hugo R. von , Müller-Berghaus G. , Graeff H.
Published: June 2019

The generalized Shwartzman reaction was produced in rabbits by two injections of endotoxin 24 hours apart. Blood was taken before the first and second injection and 1 and 4 hours after the second injection. SFMC and fibrinogen were precipitated from plasma with β-alanine. Agarose gel filtration resulted in a separation of SFMC and fibrinogen. This enabled an estimation of the SFMC concentration. The per cent amount of SFMC of the total fibrinogen content increased from 5.6±1.8 to 22.9±5.1 per cent (mean±SD) at the end of the collection period. SDS-polyacrylamide gel electrophoresis was performed of SFMC before and after reduction with mercaptoethanol. SDS and urea resistant oligomers could be demonstrated at the time when glomerular fibrin depositions were present. Chain analysis of SFMC revealed γ–γ dimers and a loss of α-chains in association with faster migratin components. This experimental observation indicates that hypercoagulability (before the second endotoxin injection) is accompanied by an elevated level of SDS and urea non resistant SFMC, whereas DIC (last sample) is evidenced by the occurrence of SDS and urea resistant SFMC containing γ—γ dimers.

(Supported by DFG, SFB 51, grant no. 2/Gra - B/6.)
Artificial Surfaces - Their Importance with Respect to Generation of Microparticles in Stored Blood
Authors: Eckert G. , Ey G. , Eisert W. G. , Stangel W.
Published: June 2019

Pulmonary microembolism is a known complication of massive blood transfusion. The development of amorphous microparticles in stored blood depends on numerous factors such as time of storage, the kind of stabilisator, and electrical potential as well as morphology of artificial surfaces of blood containers.

We have investigated the surfaces of glass and of different plastics used in Western Europe when taking and preserving blood with a scanning microscope. Clear differences have been observed among the different surfaces of plastic tubes and bags with respect to morphology. With increasing roughness there is increasing incidence of irritated thrombocytes adhearing to the surfaces, augmented adhesivness of thrombocytes, and activation of the blood coagulation system. An increase of both amount and size of microparticles as measured by a laser absorption technique, has also been observed.

Our results show that the chosen plastics, as well as the way they are produced, effects the generation of microparticles in stored blood.
Kinetics of 51Cr-Labeled Thrombocytes During Cardiopulmonary Bypass Circulation
Authors: Michiels J. M. , Lindemans J. , Jong D. S. de , Krenning-Douma E.
Published: June 2019

The decrease in number of circulating platelets during the use of cardiopulmonary bypass (C. P. B.) in cardiac surgery had been found to be larger than could be explained on the basis of hemoclilutions in the heart-lung machine (H. L. M.). It is demonstrated in seven experiments, that the radioactivity of 51Cr-labeled donor platelets, which had been transfused 24 hours prior to the operation, decrease at the same rate as the total number of platelets in the patient’s blood. It is known that many platelets adhere to the filters in the extracorporeal circuit, especially to the terminal filter in the arterial line. External monitoring over the filter area revealed that adherence of radioactive thrombocytes reached a maximum within a few minutes after complete mixing of the patient’s blood with the blood-Hemaccel® mixture in the H. L. M., and that the radioactivity remained fairly constant during the ensuing C. P. B. The disappearance rate of platelets appeared to be closely correlated with the accumulation of 51Cr-radioactivity on the filters of the H. L. M. During cardiopulmonary bypass circulation non-platelet bound radioactivity increased gradually, suggesting release of 51Cr from platelets. Irreversible A. D. P. aggregation in vitro of 51Cr-labeled platelets did not lead to release of radioactivity, but it was found after coagulation of recalcified platelet rich plasma. The results of this study indicate that during cardiopulmonary bypass circulation, platelets aggregate and adhere to the filters in the first minutes, and that subsequently platelets are damaged and/or consumed.
Haemostatic Changes Induced in Vitro by Activated Charcoal Haemoperfusion
Authors: Winchester J. F. , MacKay J. M. , Forbes C. D. , Blakely E. , Prentice C. R. M. , Kennedy A. G.
Published: June 2019

An in vitro system was devised to investigate abnormalities of haemosbasis induced in human blood by haemoperfusion through activated charcoal particles. The system consisted of identical polypropylene columns filled with either uncoated or polymer coated activated charcoal particles, blood being pumped through the system at a constant flow rate, for two hour periods.

When sodium citrate was used as the anticoagulant, the fall in platelet count was not statistically different whether coated or uncoated charcoal was used, or whether an empty (saline filled) column was used. However, coated charcoal significantly improved the changes in KCCT, PTT, OSPT, induced by haemoperfusion. Similarly the polymer coating appeared to minimise the fall in fibrinogen, factors II, V, VII, VIII, IX, X, XI, and XII.

When mucous heparin (3 μ/ml) final concentration) was used as anticoagulant, massive (80%) retention of platelets occurred with uncoated charcoal, whereas, polymer coated charcoal resulted in only 30% platelet drops.

These results suggest that haemostatic abnormalities induced by charcoal haemoperfusion are in part due to contact activation, coagulation factor adsorption, and/or heparin adsorption. Preliminary experiments with heparinised red cell free blood (PRP) have shown minimal platelet drops whether uncoated or coated charcoal was used, suggesting that red cell ADP release may play a crucial role in producing the haemostatic abnormalities, occurring with whole blood haemoperfusion.
Purification and Characterization of Activation Fragments F1 and F2 from Human Prothrombin
Authors: Ball A. P. , Fenton J. , Aronson D. L. , Franza R. B. , Young A. M.
Published: June 2019

Considerable quantities of the non-thrombin portions of human prothrombin (II) have become available as a byproduct of the large-scale production of human thrombin (Biochim. Biophys. Acta 229, 26). Components not adsorbed on CG-50 are further purified by DEAE-cellulose chromatography and gel filtration, yielding the NH2-terminal fragment (F1) and the inner fragment (F2) which are homogeneous by SDS gel electrophoresis. SDS gel electrophoresis of reduced F1 indicates variable amounts of a two-chain derivative, F1’, with one chain migrating just ahead of F1 and one just ahead of the thrombin A-chain. The F1 → F1’ conversion is catalyzed by thrombin with the creation of a new MH2-terminal threonine. Ultracentrifugal patterns of human F1 and F2 closely resemble those of the bovine fragments. NH2-terminal residues were found to be alanine (± threonine) for F1 and serine for F2. Minor deviations from the reported amino acid compositions of bovine F1 and F2 were observed, primarily in the acidic residues. Other properties include:

Immunization of rabbits with F1 gave a precipitating antibody to F1 which cross-reacts with II, but native F2 does not appear to be immunogenic. 3H-F1 is rapidly cleared from the blood of rabbits (T 1/2 20 min), with a major portion detectable in the urine.
The Relation between Staphylocoagulase Reacting Factor and Proteins Induced by Vitamin K Absence
Authors: Bas B. M. , Muller A. D. , Hemker H. G.
Published: June 2019

Five different ways of estimating prothrombin are applied to the plasma of persons receiving vitamin K antagonists, to know: the one-stage assay, the two-stage assay, the Echis Carinatus Venom assay, the coagulase-reacting factor assay and the immunological assay. The Protein Induced by Vitamin K Absence analogous to prothrombin (PIVKA-II) can be shown to be co-estimated in all but the one-stage assay. There are minor differences, however, between the other four tests. The most practical way to assess both prothrombin and PIVKA-II seems to be the coagulase-reacting factor assay. The difference between the one-stage assay and the others can be explained on basis of the new data on the role of vitamin K in prothrombin biosynthesis. The differences between the other tests are smaller and remain to be explained.
The Isolated Surface Membrane of Pig Platelets. I. Subcellular Analysis and Identification Using em Morphometry and 125I Antibody Labelling
Authors: Bullock G. R. , Baker J. R. J. , Taylor D. G.
Published: June 2019

Platelet membrane vesicles can be separated into two subfractions by centrifugation through a linear sucrose density gradient using the B14 titanium zonal rotor (Taylor and Crawford, 1974). The basis of this subfractionation is the separation of two phosphodiesterase activities. Evidence confirming that the membrane subfraction showing 8-fold enrichment of phosphodiesterase activity towards bis (p-nitrophenyl) phosphate contains surface membrane has been derived from a study of the distribution of 125I after prelabelling the whole cells with iodinated anti-platelet membrane antibody.

The circle analysis method of Williams (1969) was used to correlate the distribution of 125I with constituent organelles. This gave a p value of 0.001 and χ2 of 92.8 for the analysis and a high correlation of label associated with the plasma membrane and its elements.

References: Taylor, D. G. and Crawford, N. FEBS Letters, 41, 317-322, 1974. Williams, M. A. (1969) “Advances in optical and electron microscopy”. (R. Barer and V. E. Cosslett, eds.) 3, 219-272. Academic Press London & N.Y.
The Isolated Surface Membrane of Pig Platelets: II. Analytical Characterisation and Study of Integral Proteins
Authors: Taylor D. G. , Bullock G. R. , Crawford N.
Published: June 2019

It has been previously shown (Paper I of this series) that platelet membranes can be separated into two subfractions, one of which (MI) contains surface membrane and the second subfraction (Mil) is probably of intracellular origin. These membrane fractions have been analysed for their protein, lipid and carbohydrate contents, and both qualitative and quantitative differences are evident. There are significant differences between subfractions MI and Mil in terms of the phospholipid concentration (MI, 0.52 mg/mg protein; Mil, 0.26 mg/mg protein) and cholesterol concentration (MI, 0.21 mg/mg protein; Mil, 0.09 mg/mg protein). In addition, subfraction MI shows a higher concentration of sialic acid and hexosamine per unit weight of protein than MII However, both subfractions contain glucose, galactose, mannose and fucose and the total neutral sugar contents are approximately the same. Densitometer profiles of SDS-solubilised polypeptides separated by SDS-polacrylamide gel electrophoresis revealed prominent bands with estimated molecular weights of approximately 140,000, 90,000, 80,000, 55,000 and 45,000 present in both subfractions. The component of 45,000 daltons has been identified as actin by co-electrophoresis with purified muscle and platelet actins, and by recombination experiments with muscle myosin showing elevation of the myosin Mg2+-ATPase activity.