bluuebunny/crossref_metadata_2025 huggingface dataset:
PROTEIN C/PROTEIN S IN THE FOETAL BLOOD. ABSENCE OF BOUND PROTEINS AND C4 BINDING PROTEIN
Authors: Melissari E , Scully M F , Parker C , Nicolaides K H , Kakkar V V
Published: June 2019

Protein C, free and bound protein S and C4 binding protein levels (C4bp), were measured by electroimmunoassay in 7 pregnant women aged 22-29 years at 16-18 weeks of gestation, immediately prior to termination of pregnancy for social reasons. Protein C and protein S levels were also measured in their foetuses from blood taken through the umbilical cord. In this group of pregnant women the mean levels for protein C were 104% of normal adult mean (range 80-128%), for C4bp 100% (52-150%), for free protein S 66% (43-89%). In the foetuses the mean value for protein C was 15.3% (10.5-21%) and for free protein S 36.85% (27-47%) of the normal adult mean. Bound protein S and C4bp levels were zero. Conclusions: (1) free protein S is significantly decreased (< 2SD below the normal adult mean) in women after the first trimester of gestation whereas no change is seen in protein C concentration; (2) C4bp levels are at zero in the foetus as also are the levels of bound protein S; (3) foetal blood protein S level is approximately 2.5 times higher than protein C. Since all other vitamin K-dependent factors have been observed to be in the range of 10-20% of normal at this stage of gestation, our findings may be further proof of a non hepatic (endothelial) source of plasma protein S.
STUDY OF A SPANISH FAMILY WITH INHERITED PROTEIN S DEFICIENCY
Authors: Fontcuberta J , de los Inocentes R M , Sala N , Borrell M , Félez J
Published: June 2019

Protein S (PS) is a plasma glycoprotein that serves as a cofactor for activated protein C (PC) anticoagulant activity. Inherited PS deficiency has been found to be associated to thrombotic disease in several families. In the present study, we report on a Spanish family with type II PS deficiency.

The propositus is a 40 year-old male that was referred to our center for study after having suffered from multiples thrombotic events since he was 20 year-old. After his first episode of deep vein thrombosis (DVT) he had 4 recurrences, three of them complicated with pulmonary embolism. It should be remarked that one of the episodes occured while the patient was under oral anticoagulant treatment. The basic screening of haemostasis and hepatic function were normal for a patient that was being treated with oral anticoagulants. Functional and antigenic levels of antithrombin III, protein C and plasminogen were also normal. When total and free protein S levels (method of Comp et al.) were measured using both an electroimmunoassay and an ELISA assay ,almost indetectable levels of free protein S (between 0 and 10%) and very low levels (20%) of total plasma PS, were found. These results were also confirmed by crossed-electriimnunophoretic studies.

When the family of this patient was studied it was found that his two sons, aged 15 and 8 years, as well as one of his sisters, aged 35 years, and her daughter of 4 years, were also affected (free PS levels between 38-60% and total PS between 35 and 39%). All these members had been assymptomatic up to now and are not under oral anticoagulants.
HEMATOLOGICAL CHARACTERISTICS OF PREGNANT BLOOD AND LOCALIZATION OF THROMBOMODULIN IN HUMAN PLACENTAL VILLOUS TISSUE
Authors: Matsumoto T , Kanamaru K , Sugiyama Y , Deguchi K
Published: June 2019

Thrombomodulin(TM) is a cell surface protein found on endothelial cell that binds thrombin and increases thrombin's ability to activate protein C(PC). In the present study, we examined hematological characteristics and behavior of plasma PC level in pregnant women and localization of TM in the placental villous tissue. The results obtained are reported here. Cubital venous blood of 20 normal pregnants, 8 purperants and 60 non-pregnants. PC antigen(PC:Ag) was measured by the Laurell's technique using Assera plate-proteinC. Localization of TM was determined in such a way that the villous tissue was fixed in formalin, cut into paraffin sections, and stained by ABC method using anti-TM antibody. Coagulation-related factors of the pregnant blood, i.e. fibrinogen, FV, FVIII, FX, FXII and prekallikren showed statistically higher values compared with the control group, and AT-III showed almost simillar value to the control group or tended to decrease to scxne extent compared therewith. As for fibrinolysis-related factors, on the other hand, plasminogen, α1-antitripsin showed higher values with the progress of pregnancy, and α2-macro_ globulin showed slightly lower values in both 3rd trimester and puerperal stage. PC:Ag increased in the 2nd and 3rd trimester (p<0.01). Comparative examinations made of PC:Ag level between pre- and post-taking oral centraceptives revealed a significant (p<0.05) decrease frcm 131.0%( pre-taking) to 117.0%(post-taking). At the 11th week of pregnancy, TM was confirmed to be highly localized in syncytiotrophoblast in the villous tissue, especially in microvilli. At the 40th week, TM was also confirmed in the same site, but with weaker stainability. It was suggested that thrombosis and hemostasis-related factors were in a state of co-existing overproduction and consumption. Moreover we supposed that the existence of TM and the increase in PC might be just appropriate for the maintenance of anti-thrombogenesis in the uteroplacental circulation.
CONCENTRATION DEPENDENCE OF ACTIVATION OF ACARBOXYPROTEIN C BY THE CONTORTRIX ACTIVATOR
Authors: Exner T
Published: June 2019

The protein C activator in Southern Copperhead (Agkistrodon Contortrix Contortrix) venom was isolated by sequential chromatographies on SP�Sephadex, Con A Sepharose and hydroxylapatite. It was found to be a single chain glycoprotein with an apparent molecular weight of 36,000 and an enzymatic specificity on chromogenic substrates resembling kallikein.

This "contortrix activator" was used in a solid-phase immunochromometric assay (ICMA) for functional protein C in which heterologous antibody against protein C was passively coated onto microtitre wells and used to immoblize protein C. This was then activated, easily freed of excess activator by washing and assessed by its subsequent overnight cleavage of chromogenic substrates sensitive to activated protein C.

Correlation between protein C results obtained by ICMA and immunoradiometric assay (IRMA) on a variety of patient samples was excellent when relatively high concentrations of the venom activator was used. However with lower concentration of activator plasmas from patients deficient in vitamin K gave lower protein C values by ICMA then obtained by IRMA.

Normal protein C and "acarboxy" protein C from a patient on oral anticoagulant therapy were immuno-immobilized and studied by the ICMA technique using varying concentrations of the venom activator. The acarboxy-protein C, although completely activatable by high concentrationa of activator, was found to activate much more slowly than normal protein C at low concentrations of the contortrix activator. Thus by reducing the intensity of the activation step, the ICMA protein C results were increased in their sensitivity for functional protein C.
TWO CASES OF NEONATAL PURPURA FULMINANS HOMOZYGOUS FOR PROTEIN C DEFICIENCY IN A CHINESE FAMILY
Authors: SHEN M C , CHEN S H , LIN K S
Published: June 2019

Protein C (PC) deficiency associated with hereditary venous thromboembolic disease was first reported in 1981 and is inherited as an autosomal dominant disorder. The prevalence of heterozygous PC deficiency is estimated to be 1 to 4% in venous thrombotic diseases. The homozygous PC deficiency is even rare, and has been reported in only about 10 families througout the world. It usually presents in newborn infants as purpura fulminans or severe thrombotic disease. We herein report two newborn brothers in a Chinese family, who manifested with purpura fulminans soon after birth and died at age of 21 days and 27 days respectively. Vitamin K was administered to the second baby after birth. Both parents are not consanguineous and there were no family histories of thromboembolism on paternal and maternal sides. Blood sample was not available for specific studies in the first baby. PC antigen level by electroimmunoassay was <6% in the second baby and 49% and 60% respectively in their mother and father. Antithrombin III activity by amidolytic method was 49% in the second baby, and 90% and 97% respectively in their mother and father. Vitamin K-dependent coagulation factors and factor V were within the expected range for a newborn. Factor VIII and fibrinogen level were notably decreased. Autopsy findings of the two newborns demonstrated the similar pictures characterized by fibrin thrombi in blood vessels causing extensive hemorrhagic infarts of skin, lung, liver, kidneys, testis, urinary bladder, esophagus and brain. Our Data indicate that neonatal purpura fulminans can be familial and caused by severe homozygous PC deficiency.
PROTEIN C AND OTHER CLOTTING STUDIES IN MEMBRANOUS AND NON-MEMBRANOUS GLOMERULONEPHRITIS
Authors: Mysliwiec M , Alderson D , Poller L , Ackrill P
Published: June 2019

The occurrence of thrombosis in the nephrotic syn drome has long been known. Thrombotic complications are predominantly associated with membranous glomerulonephritis (MG). The aim of the present work was to study whether the tendency of nephrotic patients with MG to thrombotic episodes could be attributed to a hypercoagulable state. Thirty consecutive patients with the nephrotic syndrome were studied. Of these 17 suffered from MG and 13 had other forms of glomerulonephritis. The control group consisted of 10 healthy volunteers. In addition to standard coagulation assays, we studied: soluble fibrin monomer complexes (FM test, Boehringer), fibrin monomer polymerization, factor VIII:C, factor VIII:vWF, anti thrombin III (AT III) and alpha2 antiplasmin (alpha2AP) using chromogenie substrates; the levels of AT III and alpha2 AP were measured immunologically; beta thromboglobulin (BTG), platelet factor 4 and fibrinopeptide A (FPA) using radioimmunoassay kits; protein C was studied functionally and immunologically. There was a significant shortening of the prothrombin time and activated partial thromboplastin time, increase in alpha9 AP, factor V111:vWF, FPA and BTG in nephrotic patients associated with in or eases in both functional and imminclogical protein C levels and impairment of fibrin polymerization. FM test was negative in all but one of the patients. None of the coagulation tests showed a significant difference in the two nephrotic groups. High protein C and impaired polymerization may be considered as mechanisms counteracting disclosed hypercoagulability in the nephrotic syndrome.
SPECIFICITY OF INCREASED DES-GAMMA-CARBOXYPROTHROMBIN AS A MARKER OF HEPATOCELLULAR CARCINOMA AFTER VITAMIN K INJECTION
Authors: Lefrère J J , Gozin D , Soulier J P , Mavier P , Bettan L , Dhumeaux D
Published: June 2019

An elevation of des-gamma-carboxyprothrombin (DCP) has been observed in about 70 % of cases of hepatocellular carcinoma (HCC). Howewer, an increased DCP is not specific of HCC. Oral anticoagulant therapy increases the DCP level by preventing the gamma-carboxylation of prothrombin : thereafter an increased DCP can not be used as an HCC marker before three weeks have elapsed after stopping anti vitamin K therapy. Furthermore, since vitamin K is necessary for the gamma-carboxylation of vitamin K dependent factors, a vitamin K deficiency increases the DCP level long before the modification of the prothrombin time. It is thus imperative to eliminate an underlying vitamin K deficiency before attributing an increased DCP to a HCC. We used a method of DCP assay using staphylocoagulase. We studied the effect of an intravenous injection of 20 mg of vitamin K1 on DCP level in 7 patients with histologically proven HCC and in 10 patients with various disorders (5 alcoholic cirrhosis, 1 chronic hepatitis, 4 pancreatic cancer). All these 17 patients had increased DCP before vitamin K injection. In a second sampling obtained 15 days or more after injection, only the 7 patients with HCC kept increased DCP level. In patients of both categories in whom we obtained intermediary samplings, we observed that the DCP level decreased In all cases. The normalisation of the DCP level was lasting only in those patients without HCC, confirming the hypothesis of an underlying vitamin K deficiency ; this decrease was very transitory in those patients with HCC, suggesting that the elevated DCP came from a yet unexplained (but not linked to a vitamin K deficiency) mechanism. We may conclude that an increased DCP level 15 days after vitamin K injection may constitute a specific marker of HCC.
Platelet Aggregation Induced by a Synthetase-Like Substance and Phospholipase-A.
Authors: Iatridis S. G. , Iatridis P. G. , Markidou S. G. , Ragatz B. H.
Published: June 2019

Platelets contain arachidonic acid-bearing phospholipids and phospholipase-A (Phl-A); it has been suggested that exposure of these substances to each other by stimuli, such as thrombin or adenosine diphosphate (ADP), would result in liberation of arachidonic acid (AA), which is the precursor of cyclic endoperoxide; a factor causing irreversible aggregation of platelets.

By using a saline eluate of a polyurethane (SPU, -polyester elastomer of the thermoplastic type-, secured by exposing saline for two minutes to the inner surface of a polyurethane bag) we observed that platelet (human PRP) aggregation (second phase type, irreversible) could be induced by Phl-A plus SPU or AA plus SPU. These mixtures failed to aggregate aspirinized platelets. Aspirin, however, incubated for 10 minutes with SPU, Phl-A or AA did not inhibit platelet aggregation induced by the addition of the missing factor. In control experiments no aggregation was produced by SPU-saline, saline-Phl-A or saline-AA. SPU plus suboptimal concentrations of ADP or epinephrine induced an immediate in onset aggregation. Phl-A added in reversibly, by ADP, aggregated platelets produced a second phase of aggregation.

The results show that AA which is the precursor of cyclic endoperoxide could be liberated by Phl-A. The results also provide evidence that at least two enzymes (synthetase-α and synthetase-β; α is inactive whereas β is active and can be blocked by apsirin) are involved in the process of AA activation and biosynthesis of cyclic endoperoxide.

Supported by a grant from the U.S. Public Servie HL-15425.
Determination of Plasminogen by Means of a Chroniogenic Peptide Substrate
Authors: Friberger P. , Axelsson G. , Korsan-Bengtsen K.
Published: June 2019

Plasmin splits the chromogenic substrate B2-Phe-Val-Arg-pNA (S-2160, Bofors) at a relatively high rate. Standard plasmin in glycerol obtained from Nat. Inst, for Biol. Stand, and Contr., London, was tested in a system with Tris buffer of varying pH and ionic strength. The pH optimum for the reaction was found to be 7.4. Variations in ionic strength between 0.05–0.1 had insignificant influence but at higher ionic strength there was a slight inhibition. A linear relationship was found between plasmin and AOD/min. At optimum pH and a final substrate concentration of 0.2 mM 0.1 CTA unit corresponds to approximately 0.10 nkat. Purified plasminogen (AB Kabi, Stockholm, Sweden) in the concentrations 0.02–0.2 CU/ml was activated optimally with streptokinase (Kabikinase® ) in the concentrations 500–2000 IU/ml. Higher concentration gave inhibition. The activity of streptokinase activated plasminogen increased with a decreasing ionic strength. A linear relationship was found between streptokinase activated plasminogen and AOD/min. Approximately 3,000 Plong/units per ml of urokinase was needed to obtain the same activation as with optimal streptokinase concentration. The method has been used for determination of plasminogen in plasma. With final dilution of plasma in the range 1/20–1/200 activated by streptokinase (2000 IU/ml) in a system of pH 8.2, I = 0.05, a linear relationship was found between plasma dilution and AOD/min. The reproducibility in a series of tests is good (variation coefficient < 3%) and with insignificant interference by inhibitors. The determinations were easily carried out in a simple spectrometer (405 nm) and in an automatic reaction rate analyzer (LKB 8600, 410 nm).
An Artifact in Isolated Organ Perfusion Studies of Coagulation Factors
Authors: Owen G. A. , Bowie E. J. W.
Published: June 2019

Using isolated rat livers, Mattii et al. (Proc. Soc. Exper. Biol. Med. 116, 69, 1964) and Olson et al. (J. Clin. Invest 45, 690, 1966) observed generation of modest amounts of factor V. With rabbit liver-spleen combinations, Dodds and Hoyer (Br. J. Haem. 26, 497, 1974) found factor VIII emerging. We have perfused rat livers with rat blood cells in bovine albumin-Tyrode’s solution. One-stage assays for factors V, VIII, XI and XII indicated release of all 4 clotting activities within the first hour of perfusion, followed by a slow diminution. However, the same occurred if there were no liver in the perfusion system. When the rath erythrocytes were free of platelets (> 90%) and leukocytes (> 75%) before mixing with albumin-Tyrode’s, the isolated liver generated factor XII but not factors V, VIII and XI. Production of fibrinogen, prothrombin and factors VII, IX and X was not altered by removing the leukocytes and platelets.
Immunochemical Characterization of a Factor IX Inhibitor Following Anamnestic Response
Authors: Roberts H. R. , Fuller C. R. , Worden H. , Stuart J. , Reisner H. , Koehler K. , Yount W. J.
Published: June 2019

We previously characterized a human inhibitor for Factor IX in patient P.W.B, with Hemophilia B as an IgG4, λ immunoglobulin of restricted electrophoretic mobility. This restriction to a minor IgG subclass led us to characterize a second Factor IX inhibitor occurring in patient R. J. after an anamnestic response to Factor IX. On preparative zone electrophoresis the inhibitor migrated with a broad zone of mobility in the anodal portion of the γ peak and was restricted to the anodal portion of the IgG containing fractions. Gel filtration on calibrated 1.5 M sepharose columns revealed inhibitor activity in fractions corresponding to a molecular weight of 150,000. The inhibitor was further characterized by the technique of antibody neutralization using monospecific antisera to immunoglobulin classes, subclasses and light chain types in the zone of antibody excess. The inhibitor was completely neutralized by antibody to IgG whereas antisera to IgA, IgM, IgD and IgE had no effect. Neutralization was abolished by absorption of the IgG antiserum with purified IgG. Neutralization with antisera specific for light chains indicated a mixture of light chain types with an estimated ϰ/λ ratio of 6/1. Neutralization with antisera specific for IgG subclasses revealed a mixture of IgG subclasses. The Factor IX inhibitor was thus characterized as a polyclonal IgG immunoglobulin. Sepharose conjugates of R. J. globulin effect complete removal of Factor IX from normal plasma on an immunoabsorbent column and biologically active Factor IX may be eluted with 1600-fold purification.
Electroimmunoassay of Human Coagulation Factor IX in the Detection of Variants of Haemophilia B.
Authors: Orstavik K. , Osterud B.
Published: June 2019

Factor IX was quantified by an electroimmunoassay (EIA, rocket Immunoelectrophoresis), using a rabbit antiserum against human factor IX. The EIA was performed on plasma samples from 38 healthy persons. The range was 65–150% relative to a standard plasma pool, and a positive correlation was found between determinations of factor IX antigen and factor IX activity.

Seven patients with severe and six patients with moderate haemophilia B were tested for plasma cross-reacting material (CRM) by the EIA and by an antibody-neutralization test. Five of the seven patients with severe haemophilia had no detectable CRM by any of the two techniques whereas the remaining eight patients had CRM varying from low to normal values. The coefficient of correlation was 0.82 between the determinations of CRM by the two techniques.

One of the patients with severe haemophilia and normal amounts of plasma CRM showed a prolonged prothrombin time using ox brain thromboplastin (haemophilia BM). As a group, the other patients had a significantly reduced prothrombin time when compared to 20 healthy males.

Our results confirm that haemophilia B is a heterogeneous disorder and show that the EIA may be used to detect genetic variants of this disease.
Cyclical Variation in Factors VII and VIII Associated with Oral Contraception
Authors: Castaldi P. A. , McGrath K. M.
Published: June 2019

Sequential studies of the levels of coagulation factors II, VII, VIII (activity and antigen), IX, X and fibrinogen, were carried out in three females using oral contraceptive agents, and two normal controls. A striking increase in the levels of factor VII activity was seen in the second half of the menstrual cycle in all test subjects. In two test subjects, a marked cyclic increase in factor VIII activity occurred and this did not have any fixed relationship to VIII antigen levels.

The increase in factor VII activity was associated with shortening of the thrombotest in plasma tested after storage at –20 C. Activation of factor VII can be induced in vitro in 60% of oral contraceptive plasmas by incubation at 4 C for 16 hours. This cold promoted activity (CPA) also varies throughout a menstrual cycle, is independent of the prothrombin complex, being present in Al2OH3 adsorbed plasma, and is contained in the supernatant of the euglobulin fraction in CPA positive plasmas. It can induce activation of factor VII in normal plasma, but not in contact factor deficient plasma. The nature of this cold sensitive activity and its relationship to the cyclical increase seen in factor VII activity and to oestrogen induced clotting in vivo, remains to be determined.
Factor XIII Concentrate in the Long Term Management of Congenital Factor XIII Deficiency
Authors: Miloszewski K. , Losowsky M. S.
Published: June 2019

Congenital Factor XIII deficiency causes a serious and disabling bleeding diathesis with a high risk of fatal intracranial haemorrhage. Blood levels of Factor XIII of a few percent of normal are sufficient to control bleeding and the in vivo half-life of Factor XIII is long, making permanent prophylaxis practicable.

We have kept 5 patients with congenital Factor XIII deficiency free from bleeding manifestations for 3 to 10 years by regular plasma infusions. Their lives have been transformed. Plasma as a long term source of Factor XIII has disadvantages and a purified Factor XIII has been derived from human placenta (Hoechst), but its in vivo half-life was said to be only 70 hours. The only detailed report of the use of this preparation is of two injections in a single patient. We have used this preparation regularly for two years in two patients and we find that its half-life is similar to that of plasma Factor XIII i.e. 12–14 days. Both patients have remained free from bleeding manifestations or side effects.

We conclude that long term prophylaxis for congenital Factor XIII deficiency is possible and the placental Factor XIII is preferable to plasma.
The Fibrinogen-Thrombin Reaction
Authors: Hogg D. H. , Blombäck B.
Published: June 2019

The kinetics of thrombin-catalyzed release of fibrinopeptides A and B is reported. The results suggest that the N-terminal disulphide knot (N-DSK) contains the structural elements of fibrinogen which are recognized by thrombin during release of the A-peptide. In contrast release of the B-peptide requires a structure larger than N-DSK. Further, it appears that most of the structural elements of N-DSK defining the specificity of action reside in an N-terminal portion of the Aγ chain.

The mechanism of the fibrinogen-thrombin reaction is suggested as involving firstly release of the A-peptide with a consequent change in conformation in the substrate, possibly induced by polymerization, which then facilitates release of the B-peptide.
Kinetics of Homologous Urea-soluble 131I-Fibrin and 125I-Fibrinogen in Rabbits
Authors: Müller-Berghaus G. , Mahn I. , Kövecker G.
Published: June 2019

In order to study the in vivo behaviour of soluble fibrin, purified rabbit 13I-fibrinogen was clotted in the presence of EDTA (0.005 M) and aprotinin (100 units/ml), the generated clot dissolved in buffered urea (3.0 M, pH 7.4), and the formed ureasoluble 131I-fibrin injected into rabbits. The behaviour of homologous 125I-fibrinogen was simultaneously studied in the same animals. Results: The distribution volume of 131I-fibrin (44 ml/kg) as well as that of 125I-fibrinogen (45 ml/kf) was nearly identical indicating that the injected soluble fibrin was homogenously distributed in the circulation immediately after injection. The elimination of soluble fibrin from the circulating blood did not represent a monophasic exponential decay. 84% (range: 77–94%) of the injected soluble fibrin disappeared from the circulation during the first 24 hours after injection, whereas only 54% (range: 41–72%) of the injected fibrinogen disappeared during that interval. Under these experimental conditions, the T/2 of fibrinogen was 46 hours. The clearance of urea-soluble fibrin did not influence the kinetics of 13I-fibrinogen. The disappearance of soluble fibrin could not be influenced by treating the animals with aprotinin for fibrinolysis inhibition. The experiments demonstrate that soluble fibrin can be traced in the circulation and cleared from the blood by a mechanism independent of the fibrinolytic system.

(Supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.)
Immunological Cross Reaction Between Chains of Human Fibrinogen
Authors: Oårdlund B.
Published: June 2019

Employing the crossed immunoelectrophoresis technique (CIE) the antigenic composition of fibrinogen chains and heterogeneity in their electrophoretic mobility was investigated.

Highly purified chains of fibrinogen were prepared by chromatography on CM-cellulose (1). Antisera against chains were prepared by immunizing rabbits with Aα, Bβ and γ chains respectively. The CIE was performed on 10 × 10 glass plates with an 1 mm thick layer of 1% agarose in 0.04 M barbital buffer pH 8.6. The electrophoresis was run for 1 h with 12 V/cm. Four fifth of the gel was cut off and replaced with the same agarose solution containing antibodies. The second dimension electrophoresis was run for about 20 h with 2 V/cm.

Using anti Bβ chain serum a partial immunological identity was revealed between the Bβ chain and the γ chain. The Bβ chain has except for the antigenic sites in common with the γ chain additional sites that are unique for the B β chain. The results were confirmed with the anti γ chain serum which gave a strong reaction with both the γ chain and the B β chain preparations. The Aα chain did not react with γ antiserum, nor did the γ chain react with anti Aα chain serum. The immunological relation between the Aα chain and the B β chain is under investigation.

These experiments also concluded that the γ chain preparation is contaminated with a B β chain variant that has an electrophoretic mobility similar to that of the γ chain. Also the Bβ chain seemed to be impure due to contamination by an Aα chain derivative with higher electrophoretic mobility.

(1) Murano, G., Wiman, B., Blomböck, B. and Blomböck, M. : Preparation and isolation of the S-carboxymethyl derivative chains of human fibrinogen. F.E.B.S. Letters: 14, 37, 1971.
Quantitative Activator Determination in the Plasma of Patients Undergoing Fibrinolytic Treatment
Authors: Martin M. , Auel H.
Published: June 2019

A new method ist presented for estimating the activator (plasminogenstreptokinase complex) concentration in the plasma of patients undergoing streptokinase infusion. The principle of the method is based on the clotlysis time as recorded by the thromboelastograph. The test clot constituents were bovine fibrinogen, bovine plasminogen, EDTA, human plasma (with unknown activator concentrations) and thrombin. In order to obtain a standardization curve, urokinase dissolved in NaCl solution was substituted for patient’s plasma. Thus, each lysis time could easily be converted into urokinase equivalent (CTA u/ml). Streptokinase treatment using a dosage scheme of 100,000 u SK/h was monitored by quantitative activator, plasminogen, and streptokinase measurements. During streptokinase infusion an average activator concentration of 20–120 CTA u UK-equ and a SK-concentration of 3–8 u/ml were recorded. Each drop in streptokinase was accompanied by a drop in activator, and each rise in streptokinase by a rise in activator. There was a strong correlation between streptokinase and activator concentrations (correlation coefficient r = 0.9).
Effect of Beta Blockers on Human Blood Plaletets in Vivo
Authors: Zawilska K. , Komarnicki M. , Manka B.
Published: June 2019

ADP and collagen-induced platelet aggregations are diminished one hour after propanolol administration to normal subjects while adrenalin-induced aggregation and platelet factor 3 availability are not influenced. This effect of propanolol in vivo is very different from its in vitro action and is possibly related to the interaction adrenalin-ADP and collagen-ADP. Intermediary products of propanolol metabolism may also be involved in this effect.

The administration of practololol to a second group of normal subjects had no effect on platelet aggregation and on platelet factor 3 availability.
The Effect of Benzcyclan and Theophyllin on the ADP Induced Platelet Aggregation in Vitro and in Vivo
Authors: Losonczy H. , Nagy I. , Gregus Z. , Szaksz I.
Published: June 2019

Autors performed in vitro examinations on platelet-rich plasma of healthy individuals with the methods of Hovig and Born. They found that complete inhibition was caused by Benzcyclan at 6 × 10−4 Mol cc. and by Theophyllin at a cc. of 10−3. Then they studied the combined effect of the two drugs. They found that complete inhibititon occured at a concentration level at which the drugs in question could only cause a slight inhibition when applied separately. When applied in vivo the 100 mg Benzcyclan administered intravenously was found to have an inhibitive effect reaching the maximal effect within 90–120 min. only a slight inhibition was observed when 250 mg Theophyllin was administered intravenously reaching its maximal effect in 30–60 min. At simultaneous administration inhibition was first observed in 15 min. The maximal effect was reached in 60 min., but some effect could still be detected 180 min after administration and the extent of the inhibition was significantly greater than in separate application. Authors discuss this synergy observed at both in vitro and in vivo examinations, its theoretic background and therapeutic significance.
In Vivo Activity of Tieclopidine, a New Drug with Platelet Aggregation Inhibiting, Antisludge and Antithrombotic Properties
Authors: Ferrand J. C. , Aubert D. , Lacaze B. , Pepin O. , Thebault J. J.
Published: June 2019

Tieclopidine (53–32 C) belongs to a new series of synthetic compounds which have proved to inhibit significantly blood platelet aggregation in animals. Administered orally to rats, it reduces markedly the plate let aggregation induced by ADP and collagen. The activity appears 2 to 6 hours after a single dose and persists for 24 hours.

The sludge and blood stasis induced in rats by protamin sulfate injections disappear completely in five minutes after an intravenous administration of Thieclopidine. Animals pretreated orally with this compound failed to show any sludge formation.

The antithrombotic activity of Tieclopidine was demonstrated in rats carrying a dental broach implanted in the abdominal aorta. White thrombideveloped locally in the control animals, but were absent or much less severe in pretreated animals.

In man the first clinical approach has shown that at a daily dose of 1 g the inhibitory effect of Tieclopidine on ADP induced aggregation requires 24 to 48 hrs to become significant and reaches a plateau after 5 or 6 days of treatment.
Fitzgerald Factor and Surface-Mediated Reactions
Authors: Saito H. , Ratnoff O. D. , Waldmann R. , Abraham J. P.
Published: June 2019

The kaolin-activated partial thromboplastin time (PTT) was abnormally long in an asymptomatic 71 year old man whose plasma contained normal amounts of recognized clotting factors except Fletcher factor, the titer of which was 10–15% of normal. Prolonged incubation with kaolin before recalcification did not shorten the PTT. The defect resided after activation of Hageman factor (F XII) and Fletcher factor and before participation of activated PTA (F XI). Other surface-mediated reactions – generation of kinins, evolution of PF/Dil, augmentation of Factor VII activity and generation of fibrinolytic activity in acidified, diluted plasma exposed to kaolin – were all impaired, the last despite the presence of adequate amounts of urokinase-activable plasminogen and HF-cofactor. A corrective fraction of normal plasma was partially purified by chromatography on QAE-Sephadex, SP-Sephadex and Sephadex G-150; a similar fraction of the patient’s plasma was inert. The correction fraction had a sedimentation constant of 5.0 and an apparent MW by gel filtration of 160,000. The corrective agent – named Fitzgerald factor after the patient’s surname – shortened the patient’s PTT and, in small amounts, induced partial correction of impaired kaolin-induced fibrinolysis. The site of origin of Fitzgerald factor is not known, but its titer was diminished in hepatic cirrhosis, but not in coumadin-treated cases, renal failure or rheumatoid arthritis. Low titers were observed in patients undergoing disseminated intravascular coagulation. The hereditary nature of Fitzgerald trait is not yet established.

Activation of PTA and augmentation of Factor VII activity require the presence of activated HF and Fletcher factor, but additional factor or factors are needed. The studies described suggest that one additional factor needed for surface-mediated reactions is Fitzgerald factor.
The Treatment of Asphyxiated Newborn with Dic by an Alpha Blocking Agent
Authors: Yamada K. , Shirahata A. , Meguro T.
Published: June 2019

The alpha blocking agent was used in the cases of newborn infants with respiratory distress syndrome (RDS) in which DIC developed. The cases of RDS included idiopathic respiratory distress syndrome, massive aspiration syndrome, atelectasis, etc. To those infants, 1 mg per kg of phenoxybenzamine (POB) was infused every 24 hours. In some of the cases, the exchange transfusion was combined with the administration of POB. After the infusion of POB, almost all of abnormal findings in coagulation studies improved remarkably as did the clinical conditions of DIC, except for the cases of sever stage.

In order to analyse our clinical results, a set of animal experiment was carried out. In this study, the rabbits which were rendered asphyxic were infused with POB and the pharmacological effects of this agent on asphyxia were examined from the aspects of coagulation and platelet functions.
ICSH-ISTH International Anticoagulant Control Study. I. Background and Co-ordination
Authors: Schmidt R. M.
Published: June 2019

Numerous thromboplastins and control plasmas are now available for prothrombin time tests in the control of oral anticoagulants. Quality Control schemes are running in various countries, and a number of attempts are being made to standardize reagents. In addition, the National Institute for Biological Standards and Control (NIBS & C), London, holds for distribution five lyophylized thromboplastins which could serve as a primary and four secondary reference materials. Broadly-based data on the relative performance of the more widely-used reagents are therefore urgently needed, and the International Committee for Standards in Haematology, co-sponsored by the International Society on Thrombosis and Haemostasis, has established an Expert Panel on Oral Anticoagulant Control. An international collaborative study was conducted in the autumn of 1974 involving some 180 laboratories in 24 countries; 10 widely used thromboplastins, the NIBS & C materials and three series of lyophylized control plasmas representing different degrees of anticoagulant effect were tested in parallel with local working thromboplastins and fresh plasma samples from patients on anticoagulant treatment.

The background, purpose and conduct of the trial will be described.
Progress in Proficiency Testing of the One-Stage Prothrombin time in the Federal Republic of Germany
Authors: Beeser H. , Fischer J. , Merten R
Published: June 2019

With regard to the small range of the therapeutic prothrombin complex levels the treatment with oral anticoagulants needs reliable laboratory controls. The results of the one-stage prothrombin time generally used as control method are however known to be poorly comparable between different laboratories. This difficulty arises particularly from the use of numerous types of thromboplastins, insufficient reference materials and calibration curves.

In the Federal Republic of Germany we three times a year conduct quality control trials of the one-stage prothrombin time according to the Bundesärztekammer (BAK) directions on external quality control of clinical laboratory methods. Participating laboratories are asked to determine prothrombin times of normal and abnormal freeze dried plasma samples using their own thromboplastin reagents and methods. For reasons remarked above a uniform evaluation of the results of all laboratories is impossible.

We can state: 1. Wide variation of the seconds for prothrombin times, even if identical thromboplastins are used. But the increasing use of mechanical devices brings along an obvious improvement. 2. The variability of the percentage results tends to decrease probably due to better calibration of the methods. 3. Prothrombin time ratios show a good comparability for each type of thromboplastin. Grouping of qualitively similiar thromboplastins has proved to be practicable. 4. There is found an increasing conformity of all mentioned parameters between expert labs and other labs.
Inherited Antithrombin III Deficiency and Thrombo-Embolism
Authors: Egeberg O.
Published: June 2019

Thrombophilia due to inherited deficiency of blood antithrombin III (AT III, heparin cofactor, anticonvertin) in a Norwegian family was published 1965, Thromb. D. h. 13, 516 & 14, 473. Only a few families with this defect have since then been described in different countries. In another Norwegian family, two sisters, age 42 and 30, and a brother, 35, have had episodes of venous thrombosis and pulmonary embolism from the age of 24–29. Their father suffered from thrombosis and died at 67. The two sisters have blood AT III level about half of normal average, measured with a two-stage coagulation assay. Data from both families are compatible with an autosomal dominant inheritance of the plasma protein deficiency. Venous thrombosis in the families is remarkably often complicated with embolizations; this might also relate to an inadequate platelet function. Platelet aggregation time of PRP with added thrombin or ADP was found prolonged. In coumarin treatment of the patients, AT III assaying gave increased levels.
Factor XIII, Fibrin and Collagen
Authors: Nyman D. , Duckert F.
Published: June 2019

The significance of factor XIII (fibrin stabilizing factor) in hemostasis and wound healing is well known. The significance of fibrincrosslinking has, however, not been unequivocally demonstrated. As an additional hypothesis we postulated that factor XIII could bind fibrin to collagen by transamidination. To test this hypothesis:

1) Normal plasma or F XIII deficient plasma, collagen or buffer, Ca++ and thrombin were incubated at 37°. The clots were washed and dissolved in SDS, urea and 2-mercap-thanol. Electrophoresis on 7% SDS-polyacrylamide showed disappearance of the γ-dimer in the clot of normal plasma in presence of collagen. N-acetylation of the collagen abolished the disappearance of the γ-dimer.

2) To normal plasma, F XIII free plasma or normal plasma with EDTA, 125I-fibrinogen was added. The plasma-collagen mixtures were incubated with thrombin and Ca++, without Ca++ for the normal EDTA-plasma. The clots were solubilized as described. After extensive washing the radioactivity retained on the collagen was measured. The collagen from F XIII free clots had 23-47% and that from normal EDTA-plasma 23-46% of the radioactivity of the collagen from normal recalcified plasma clots.

These results are interpreted as demonstration of binding of fibrin to collagen by F XIII catalyzed transamidination. The importance of this binding for normal hemostasis and wound healing is emphasized.
The Reaction between Prothombin and Staphylocoagulase
Authors: Hemker H. C. , Muller A. D. , Bas B. M.
Published: June 2019

The reaction between prothrombin and staphylocoagulase is studied.

a. Optimal amounts of the active reaction product (coagulase-thrombin) are found when equimolar amounts of prothrombin and staphylocoagulase are added together.

b. The molecular weight of coagulase-thrombin equals the sum of the molecular weights of staphylocoagulase and prothrombin, both when estimated by gelfiltration and by SDS-polyacrylamide gel electrophoresis.

c. The amino acid composition of coagulase-thrombin is not in contrast with the sum of the amino acid compositions of staphylocoagulase and prothrombin.

d. In a preparation of coagulase-thrombin the N-terminal amino acids are those of prothrombin (alanine) and staphylocoagulase (aspartic acid).

e. An antibody against coagulase-thrombin precipitates both prothrombin and staphylocoagulase but not thrombin.

f. It is concluded that the thrombin acitivity in coagulase-thrombin is a result from a stoichiometric reaction between one molecule of prothrombin and one molecule of staphylocoagulase and that limited proteolysis does not play a role in this mechanism.
Effects of Aggregating Agents, Cytochalasin B (CB) and Neuraminidase (N) on the Ultrastructure of Freeze Dried (FD) Human blood Platelets
Authors: Davis R. B.
Published: June 2019

Frozen and freeze dried human blood platelets remain intact morphologically when preserved by cryoprotective agents. These studies have investigated effects of 1) the release reaction, 2) discoid stabilization by CB, and 3) N on platelet morphology in FD specimens. Platelets were collected in 1/10 volume of acid citrate and platelet rich plasma (PRP) obtained by centrifugation. Aggregating agents (adenosine diphosphate, 2 × 10-6 M, epinephrine, 5 × 10-5 M, collagen, 30 μg/ml, thrombin 0.2 U/ml) CB (25 μg/ml), trypsin (3 mgs/ml), and N (20 U/ml), then cryoprotective agents, were added. Platelets were FD and the ultrastructure examined. Aggregating agents were associated with 1 ) the appearance of amorphous electron dense material within platelets extending via channels to the exterior; 2) membranous complexes contiguous to plasma membranes; 3) numerous organelles adjacent to the plasma membrane. Platelets in artificial thrombi also showed homogeneous electron opaque areas and membrane rich surface complexes. CB caused vacuolization and previously reported concentric membranous structures were noted in trypsinized platelets. N did not prevent interplatelet bridging. In conclusion, aggregated FD platelets differ from platelets fixed by traditional means, providing morphologic support that platelet organelles and membrane systems relate structurally to the platelet exterior as well as the canalicular system to provide a catalytic lipoprotein surface during the release reaction.
Role of Trisodium Citrate in the Unclottability by Thrombin of Fibrinogen Metz
Authors: Soria C. , Soria J. , Samama M. , Poirot E. , Kling G.
Published: June 2019

In a case of homozygous dysfibrinogenemia, the whole blood clotting time was moderately prolonged, while the thrombin clotting time was infinite, whatever dose or nature of thrombin used. Besides, the bleeding syndrome in this case was very weak.

We observed also that only after trisodium citrate addition to purified fibrinogen, the abnormal fibrinogen became unclottable by thrombin even after addition of calcium chloride, since without trisodium citrate thrombin time was only prolonged.

By immunoelectrophoresis and by isofocusing in the presence or in the absence of trisodium citrate, we therefore undertook to show that trisodium citrate reacts more strongly with the abnormal fibrinogen than with normal one. Thus, trisodium citrate conferring a negative charge to the pathological molecule, the abnormal fibrinogen became resistant to clotting with thrombin. Protamine sulfate, by positiving the charges of fibrinogen, partially corrects the defect in fibrin formation.
Release of Platelet Factor 4 (PF 4) During Repeatedly Induced Platelet Aggregation in Vivo
Authors: Vaage J. , Gjesdal K.
Published: June 2019

After induced intravascular platelet aggregation a pulmonary vasoconstriction occurs due to the release of vasoactive substances from platelets (Acta physiol. scand. 92, 546-556, 1974). PF 4 has been suggested to be a marker of the platelet release reaction. In the present experiments the release of PF 4 and the vasoactive effects of induced platelet aggregation were compared in cats.

Successive i. v. infusions of either ADP or collagen were given to induce platelet aggregation. The plasma level of PF 4 was measured by electroimmuno assay immediately before each infusion and at the maximal ensuing lung response. The initial infusion of either ADP or collagen increased PF 4 in plasma by about 25%. After several ADP-infusions neither lung responses nor PF 4 release could be elicited by further infusions, although reversible aggregation of platelets was still induced. At this stage collagen infusions gave profound lung responses, but no release of PF 4. Another group of animals were given 3 successive collagen infusions. The lung responses were similar after each infusion, but the release of PF 4 became gradually reduced.

These data suggest that the release of PF 4 from platelets is dissociated from that of the vasoactive agents.
On the Action of Intravenously Applied Acetylsalicylic Acid (ASA) on Platelet Functions, a Kinetic Study
Authors: Loew D. , Vinazzer H.
Published: June 2019

There is general agreement that ASA inhibits platelet aggregation, adhesion and release reactions when given orally. In the present study, 10 individuals received a single intravenous dose of 500 mg ASA to examine the kinetics of the influence on platelet functions. Blood was drawn prior to ASA and at intervals between 2 minutes and 72 hours after injection.

Collagen induced platelet aggregation as well as PF 3 and PF 4 release started to decrease 2 minutes after ASA and reached a minimum after 1 hour. A full ASA effect could still be observed after 24 hours though ASA had disappeared from plasma by that time. Simultaneously, in-vitro examinations with ASA were carried out. ASA was added to fresh platelet rich plasma in a concentration correspondent to the in-vivo dose. The inhibition of platelet aggregation and PF 4 release had a lag time of 1 hour and was considerably less distinct than in vivo. No inhibition of PF 3 release could be observed. The results demonstrate a rapid action of ASA when given intravenously while the in-vitro effect is much less distinct. A probable explanation is a direct effect of ASA on the platelet membrane. The enhancement in vivo is supposed to be caused by splitting of the acetylic group from ASA.