bluuebunny/crossref_metadata_2025 huggingface dataset:
A Simplified Thromboplastin Calibration Procedure for Standardization of Anticoagulant Control
Authors: Loeliger E. A , van Halem-Visser L. P
Published: July 2018

In thromboplastin calibration, the possibility of replacing fresh individual plasmas by pooled fresh plasmas (used immediately after preparation or after storage at —25° C) or by pooled lyophilized plasmas has been investigated. It was found that the thromboplastin sensitivity ratios (TSR’s) assessed with pooled fresh plasmas show no divergence from those obtained with the original Biggs/Denson calibration procedure. With pooled lyophilized plasmas a small divergence can be found in particular with so-called PIVKA-insensitive rabbit-tissue thromboplastins. Since, in contrast to the original procedure, only a limited number of determinations with pooled fresh or lyophilized plasmas are needed to obtain an accurate TSR value, the modification substantially simplifies thromboplastin calibration. Moreover, the technique using lyophilized plasmas can be applied in laboratories where an insufficient number of patients are available or none at all. However, as long as pooled lyophilized plasmas display a more or less pronounced activation, it might be preferable to restrict calibration to qualified expert laboratories, i.e. national reference laboratories.

The merits of pooled lyophilized plasma are those of good stability even under the condition of accelerated degradation. They might be just as useful as thromboplastins for use as (long-term) reference material in the standardization and quality control program of manufacturers of thromboplastins, and for laboratorians preparing their own thromboplastin for the anticoagulant control.

Conditions for the preparation and use of lyophilized reference plasmas have been worked out and are presented.
Haemophilia A in a Patient with Testicular Feminization
Authors: Andrejev Yu. N , Korenevskaya M. I , Rutberg R. A , Dukarevitch M. Z , Pokrovskiy P. I , Tokarev Yu. N
Published: July 2018

A case of typical haemophilia A in phenotypically “hairless” woman aged 18 with complete testicular feminization (primary amenorrhea, absence of palpated gonads, negative sex chromatin, fluorescence of Y chromosome in interphase nuclei, caryotype 46, XY by common and fluorescent methods) is reported in this paper. Both diseases are of familial character with transmission through female line. The propositus’ father did not suffer from haemophilia. Signs of Morris syndrome (delay of menses, absence of secondary hair) are present in the propositus’ mother and sister. The propositus’ niece, the daughter of her sister, also suffered from testicular feminization, but without any haemophilia, probably due to the crossing-over. The analysis of the pedigree gives no information in favour of sex-linked or autosome-dominant sex limited inheritance of testicular feminization. Haemophilia A in the propositus is characterized by the very low factor-VIII level – 1.66%, and typical joints bleedings since 3 years. At the age of 18 the patient had major surgery for a haemophilic thigh pseudotumor. There was rapid consolidation of the fracture and contracture was erradicated. In spite of severe factor VIII deficiency the course of haemophilia in the propositus is clinically milder than in male haemophiliacs in her family and in the general population.

The similar findings were reviewed in literature including the case of hemyzygous haemophilia with testicular feminization and described women-homozygotes. The suggestion is made that the course of haemophilia in the propositus is affected by the female phenotype. The psychological status of the patient and the possibility of professional rehabilitation by surgical treatment is discussed. The need for detailed cytogenetical examination of the persons with “female” haemophilia or the other sex-linked diseases to establish the correct diagnosis and for exclusion of the possible mosaicism is discussed.
The Effect of Oral Contraceptive Estrogen on Blood Coagulation and Fibrinolysis
Authors: Hedlin Anne M
Published: July 2018

The changes in fibrinogen, antithrombin activity, spontaneous fibrinolytic activity, plasminogen and fibrinogen degradation products were studied in women, aged 18 to 30 years, who were using oral contraceptive preparations. Blood samples were obtained before use, as well as during the first month and again after several months of use. The results were divided into three groups based on the antiovulatory activity of the estrogen component of the oral contraceptive, ie. Mestranol, 50 μg, Mestranol 80 μg or Ethinyl estradiol 50 μg and Mestranol 100 μg. In each of the three groups there was a similar pattern of increase in spontaneous fibrinolytic activity and plasminogen but decrease in antithrombin levels during the monthly cycles. The fibrinogen level was increased in the first month of oral contraceptive use in the three groups but after several months of use the pattern of change seen in the first month was repeated with the lower estrogen compounds, but in the group using the preparation with 100 μg Mestranol, the fibrinogen level returned to that of the control month. The level of fibrinogen degradation products remained unchanged.
Lipids as Triggering Factors in Thrombosis
Authors: Nordøy Arne
Published: July 2018

An association has been established between acute and more persistent changes in lipid metabolism as reflected in plasma lipids, and platelet lipid metabolism. Platelet function is affected, particularly the activity and availability of platelet factor 3, however, also other changes making the platelets more sensitive to aggregating substances without interfering with the lipid part of platelet factor 3, have been documented. Experimental studies have demonstrated an increased tendency to thrombosis in animals given a diet with a high fat content with a high ratio of saturated to polyunsaturated fatty acids. Studies in man have mainly established a connection between dietary fats, plasma lipid abnormalities and frequency of coronary heart disease and clinical studies more directly relating thrombosis to lipid metabolism is highly warranted. Many open questions remain to be answered. Probably most relevant would be to understand how the antithrombotic mechanisms in the body are affected by changes in lipid metabolism. Even if thrombotic lesions are very common events in the western world our knowledge based on laboratory and experimental studies should indicate a much higher incidence, solely based on interactions between lipids and platelets in subjects exposed to our dietary habits and our way of life.
Clinical Trials in Thrombosis: Secondary Prevention of Myocardial Infarction
Authors: Klimt Christian R , Doub P. H , Doub Nancy H
Published: July 2018

Numerous in vivo and in vitro experiments, investigating the inhibition of platelet aggregation and the prevention of experimentally-induced thrombosis, suggest that anti-platelet drugs, such as aspirin or the combination of aspirin and dipyridamole or sulfinpyrazone, may be effective anti-thrombotic agents in man. Since 1971, seven randomized prospective trials and two case-control studies have been referenced in the literature or are currently being conducted, which evaluate the effects of aspirin, sulfinpyrazone, or dipyridamole in combination with aspirin in the secondary prevention of myocardial infarction. A critical review of these trials indicates a range of evidence from no difference to a favorable trend that antiplatelet drugs may serve as anti-thrombotic agents in man. To date, a definitive answer concerning the therapeutic effects of these drugs in the secondary prevention of coronary heart disease is not available.
Evidence for an Ester Bond between Thrombin and Heparin Cofactor
Authors: Owen Whyte G , Penick George D , Yoder Elizabeth , Poole Barbara L
Published: July 2018

Heparin cofactor, a thrombin inhibitor, is purified from human plasma by affinity chromatography on heparin-agarose. The nature of the binding between thrombin and the inhibitor is studied by treatment of the complex with 6 M guanidinium chloride, hydroxylamine, and dilute alkali. The complex is not dissociated during gel chromatography in 6 M guanidinium chloride. This result supports an earlier proposal that formation of the complex includes the formation of a covalent bond. Treatment of dodecyl sulfate-denatured complex with hydroxylamine results in dissociation of the complex to yield free thrombin and heparin cofactor. Hydroxylamine does not dissociate the complex unless it is denatured. The complex is also dissociated in dilute sodium hydroxide (pH 12) solutions. These results indicate that the covalent bond between thrombin and the inhibitor is a carboxylic ester.
Biological Control of Factor VII
Authors: Nemerson Yale
Published: July 2018

The tissue factor pathway is initiated by factor VII in the presence of tissue factor. The first proteolytic reaction involves cleavage of factor X by factor VII. Activated factor X, the product of this reaction, can activate factor VII by cleavage of a specific bond. The apparent coagulant activity of factor VII then rises about 60-fold. Activated factor X can also inactivate factor VII by catalyzing the cleavage of a second bond which results in a three chain molecule. Fragments of Hageman factor and perhaps kallikrein can also activate factor VII. Hageman factor, however, does not catalyze the inactivating cleavage of factor VII at a significant rate.

Recent data showing that the tissue factor pathway can activate the intrinsic system are discussed. We have shown that activated factor X, which can be generated by the tissue factor pathway, can feed back and activate factor IX in a calcium and phospholipid requiring reaction.
Specificity of Antisera to Human Fibrinopeptide A Used in Clinical Fibrinopeptide A Assays
Authors: Nossel Hymie L , Butler Vincent P , Wilner George D , Canfield Robert E , Harfenist Elizabeth J
Published: July 2018

Distinction between fibrinopeptide A (FPA) and larger polypeptides containing the FPA sequence is critical for the interpretation of clinical results with FPA immunoassay methods. Therefore, the immunochemical reactivity of 14 rabbit anti-FPA sera with six different FPA containing antigens was studied in detail. Antigens tested included: fibrinogen; fragment E of fibrinogen; the amino-terminal disulfide knot of fibrinogen; Aα 1 (Ala)-51 (Met); Aα l(Ala)-23(Arg); and, FPA. Synthetic partial sequences of FPA were also tested. The 14 FPA-specific antisera were divided into 3 distinct categories with: I, FPA immuno-reactivity of larger polypeptides containing FPA approximately 1/100 of FPA on a molar basis; II, FPA immunoreactivity of the larger polypeptides intermediate between I and III; and III, FPA immunoreactivity of the larger polypeptides approximately equal to that of FPA on a molar basis. The antigenic determinants of a category I antiserum (R 2) are included in Aα 7(Asp)-16(Arg) with Asp(7), Phe(8) and Arg(16) being essential. When attached to FPA, the sequence Gly(17)-Arg(23) decreases the immunoreactivity of FPA with category I antisera 100-fold.

The practical consequence of these findings is that, when category III antisera are employed, both FPA and larger FPA-containing polypeptides are equally immunoreactive. Since thrombin treatment of the larger polypeptides does not alter their immunoreactivity, category III antisera cannot discriminate between FPA and the larger polypeptides. On the other hand, with category I antisera, although the immunoreactivity of FPA itself is unaltered by thrombin treatment, larger polypeptides [e.g., Aα l(Ala)-23(Arg)] show a 100-fold increase in immunoreactivity following thrombin treatment and thus can readily be identified and separately quantitated. It is concluded that antisera with the specificity of category I are essential for the specific and accurate measurement of FPA, and for its distinction from larger FPA-containing polypeptides, in clinical plasma samples.
Immunologie Studies in von Willebrand’s Disease
Authors: Sultan Y , Simeon J , Maisonneuve P , Caen J. P
Published: July 2018

Two patients with a severe von Willebrand’s disease characterized by no detectable factor VIII related antigen in their plasma received transfusions of cryoprecipitate. The bleeding time was corrected for a short period of time and returned to its pretransfusional value although the other parameters of the disease were still corrected. Electrophoretic and immunologic properties of factor VIII related antigen infused were determined serially after transfusion. Modifications of these properties occurred progressively after transfusion. The half disappearance time of F. VIIIR. A. was determined and found to be considerably shorter than in hemophilic recipients. This study suggests an alteration in vivo of F. VIIIR. A. infused into von Willebrand recipients.
Metabolie Properties of Human Platelet Membranes
Authors: Kaulen Hermann Dietrich , Gross Rudolf
Published: July 2018

Washed human platelets have been labeled with either 32Pi or glycerol- 1-14C and the distribution of the label in the phospholipids determined. 32Pi was introduced primarily into polyphosphoinositides, i. e. di- and triphosphoinositide, whereas the label from glycerol which indicates de novo synthesis of lipid molecules did not appear in these phospholipids. In the course of thrombin-induced aggregation and release the phosphate incorporation into phos-phatic acid, di- and triphosphoinositide was rapidly stimulated in parallel to the platelet reaction. The incorporation of glycerol did not change under the same conditions.

It is concluded that phosphoinositides with rapid incorporation of phosphate groups are not as rapidly synthesized de novo and presumably form a separate phospholipid pool in the platelets. Only the phosphorylating reactions are stimulated by the thrombin aggregation. The necessary enzymes for these reactions, namely diglyceride kinase, phosphatidylinositol kinase, and phosphatidylinositol-phosphate kinase all can be shown to be associated with a well characterized platelet membrane fraction.
Studies on the Thromboplastic Effect of Human Plasma Lipoproteins
Authors: Sandberg Helena , Andersson Lars-Olov
Published: July 2018

Human plasma lipoprotein fractions were prepared by flotation in the ultracentrifuge. Addition of these fractions to platelet-rich, platelet-poor and platelet-free plasma affected the partial thromboplastin and Stypven clotting times to various degrees. Addition of high density lipoprotein (HDL) to platelet-poor and platelet-free plasma shortened both the partial thromboplastin and the Stypven time, whereas addition of low density lipoprotein and very low density lipoprotein (LDL + VLDL) fractions only shortened the Stypven time. The additions had little or no effect in platelet-rich plasma.

Experiments involving the addition of anti-HDL antibodies to plasmas with different platelet contents and measuring of clotting times produced results that were in good agreement with those noted when lipoprotein was added. The relation between structure and the clot-promoting activity of various phospholipid components is discussed.
Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins
Authors: Steiner M
Published: July 2018

The effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.
Effects of Dextran on the Molecular Structure and Tensile Behaviour of Human Fibrin
Authors: Dhall T. Z , Bryce W. A. J , Dhall D. P
Published: July 2018

Characteristic changes induced by dextran during the conversion of fibrinogen to fibrin have previously been shown to be associated with profound alterations in morphology of fibrin. However, whether dextran is incorporated into the fibrin molecule and whether morphological changes are associated with alterations in mechanical behaviour of formed fibrin was unclear. The investigations described show that the fibrin made in the presence of dextran has a shortened syneresis time, a lowered modulus of elasticity, an increased elongation and diminished ultimate strength at break. The molecular composition of fibrin clots remains unaltered despite the altered mechanical properties and morphological changes. Furthermore, dextran is not incorporated into the fibrin structure in any appreciable quantity. It is suggested that these several effects of dextran on clot morphology, tensile behaviour and kinetics of fibrin formation arise from increased forces of attraction between fibrin molecules such that fibrin chains are held together by weak secondary cross-links rather than by stronger primary cross-links which are hidden within the thicker fibrin chain bundles.

Recently Muzaffar et al. (1972 a, c) showed that the addition of small amounts of dextran in vitro to purified fibrinogen solution as well as platelet – poor plasma induces characteristic alterations in the kinetics of fibrin formation on account of accelerated polymerization of fibrin monomer. Further, fibrin made under such conditions was found to differ morphologically from control fibrin (Muzaffar et al. 1972 b) and to show an increased coarseness of individual fibrin fibre on electron microscopy together with an associated looseness of fibrin mesh. The degree of abnormality in fibrin morphology was found to be determined by the final dextran concentration. More significantly, dextran was demonstrated to induce similar changes in the kinetics of fibrin formation as well as in fibrin morphology of clots made from plasma after the infusion of clinical dextran fractions in pharmacological dosage in man. However, it is not clear whether dextran-induced structural alterations in fibrin are indicative ofbasic differences in its molecular composition nor whether these changes are associated with alterations in the mechanical strength of fibrin. Clearly these are fundamental questions with respect to the pharmacological effects of dextran in relationship to its clinically well documented antithrombotic activity (Lambie et al. 1970). We have investigated both these questions and report here our principal findings.
Chemorheological Studies of the Effect of Heparin on the Course of Coagulation
Authors: Overholser K. A , Baysinger C. B , Harris T. R , Deveau T
Published: July 2018

The influence of sodium heparin on viscoelastic change during coagulation was determined in vitro for whole blood samples from ten normal subjects at heparin concentrations ranging from 0 to 1.45 units/(ml whole blood). A four-parameter chemorheological model was used to describe the time course of coagulation as measured by the Weissenberg Rheogoniometer. One parameter compares closely with the whole blood activated partial thromboplastin time, while the other three may be related to the chemical kinetics of clotting.

The chemorheological model and experimental techniques were then tested in a dog preparation. It was found that rheological measurements are more self-consistent than either thrombelastography or the activated partial thromboplastin time for the assay of in vivo heparin in two dogs.
Management of Hemophilia in Spain
Authors: Martin-Villar J , Ortega F , Magallon M
Published: July 2018

Results are presented of a nation-wide inquiry into the management of hemophilia in Spain. It is calculated that there are 1,172 hemophilic patients in the country. A survey has been made of the therapeutic materials (prepared commercially or by hospitals) used in 1974 and their availability in the various hospitals of the Social Security Service. It is estimated that an average of 10,708 units of deficient factor were used per patient in this year. The number of patients on home therapy is 1.2% of the total. All cost of hemophilia treatment is covered by the Social Security. Comments are made on progress during the last five years.
Correlation between Baseline Plasminogen Activator Inhibitor Levels and Clinical Outcome during Therapy with Tissue Plasminogen Activator for Acute Myocardial Infarction
Authors: Sane David C , Stump David C , Topol Eric J , Sigmon Kristina N , Kereiakes Dean J , George Barry S , Mantell Susan J , Macy Elizabeth , Collen Désiré , Califf Robert M
Published: July 2018

Baseline plasminogen activator inhibitor (PAI) levels were examined for their influence on the responses to thrombolysis with recombinant tissue plasminogen activator (rt-PA) administered for acute myocardial infarction during the Thrombolysis and Myocardial Infarction (TAMI)-I study. Baseline PAI activity was 19 ± 21 IU/ml (normal <5 IU/ml) and baseline PAI-1 antigen 54 ± 53 ng/ml (normal 27 ± 16 ng/ml), confirming previous findings of elevated PAI levels during acute myocardial infarction. Among clinical outcomes, lower PAI-1 antigen levels correlated weakly with greater patency at the 90 min angiogram. Thus, high baseline plasma PAI-1 levels may be detrimental to reperfusion with t-PA. There was no correlation with other major in-hospital clinical outcomes including reocclusion at the 7-10 day angiogram, survival to discharge, or bleeding. During the follow up period of 2.0 ± 0.4 years, no relationship between baseline PAI levels and post-discharge reinfarction was observed.
Synergism between Tissue-Type Plasminogen Activator and a Genetically Engineered Variant Lacking the Finger Domain, the Growth Factor Domain and the First Kringle Domain
Authors: Mattson C , Wikström K , Sterky C , Pohl G
Published: July 2018

A modified variant of human tissue-type plasminogen activator (t-PA) lacking the finger domain (F), the growth factor domain (G) and the first kringle domain (K1), has an extended plasma half-life in vivo, compared to that of t-PA. When the variant (denoted K2P) was tested in vitro for its ability to lyse human plasma clots we found that the activity was characterized by a time lag phase and a sigmoidal dose-response curve. However, an attenuation of the lag phase in vitro was observed both when K2P was mixed with t-PA in a w/w ratio of 4 : 1 and when K2P was allowed to lyse a clot that had been pre-exposed to t-PA i.e. submitted to a limited plasmic digestion. Dosis that in vitro caused 50% lysis within 6 h were calculated from individual dose-response curves and were for K2P, t-PA and K2P/t-PA (4 : 1 w/w) 540 ng/ml, 360 ng/ml and 310 ng/ml, respectively. These results indicated a synergistic effect between K2P and t-PA. However, the data from individual dose-response curves showed that the effect of the K2P/t-PA mixture never was better than that of t-PA alone, and the synergistic effect in vitro is therefore considered to be of limited use. The thrombolytic activity in vivo was evaluated in a rabbit jugular vein thrombus model. Despite the lag phase observed in vitro, K2P was approximately 3 times as effective as t-PA in vivo (bolus injection). The thrombolytic effect of K2P was further potentiated when it was administred together with a small amount of t-PA (4 : 1 w/w). This potentiation in vivo was, in contrast to the effect in vitro, a useful synergistic effect as the dose-response curve for the K2P/t-PA mixture was steeper than that of t-PA and K2P alone. Doses that caused 50% lysis within 3 h were for t-PA, K2P and K2P/t-PA 1.28 mg/kg, 0.56 mg/kg and 0.35 mg/kg, respectively.
Effect of Picotamide on Platelet Aggregation and on Thromboxane A2 Production In Vivo
Authors: Minuz P , Lechi C , Arosio E , Guzzo P , Zannoni M , Lauciello C , Lechi A
Published: July 2018

Effects of picotamide (900 mg in 3 oral administrations for 7 days) on ex vivo and in vivo platelet T×A2 production and on platelet aggregation wpre evaluated in 8 patients with peripheral arteriopathy and in 8 normal subjects. Picotamide significantly reduced ADP-induced platelet aggregation, but had no effect on
that induced by arachidonic acid or the thromboxane analogue U46619. Though ex vivo platelet T×A2 production (T×B2 concentration after arachidonic-acid-induced aggregation) was reduced from 946 ± 141 (mean ± SD) to 285 ± 91 ng/ml in controls and from 1515 ± 673 to 732 ± 420 ng/ml in patients with arteriopathy, there was no effect on urinary excretion of 2,3-dinor-T×B2 (in vivo indicator of platelet T×A2 production), or on in vivo PGI2 production (urinary excretion of 6-keto-PGF1α and 2,3-dinor-6-keto-PGF1α). In the same subjects, single-dose aspirin reduced ex vivo T×B2 production by at least 98% and 2,3-dinor-T×B2
excretion from 116.7 ± 61.4 to 32.6 ± 17.0 nglg creatinine in control subjects, and from 156.3 ± 66.1 to 59.1 ± 19.2 ng/g creatinine in patients with peripheral arteriopathy. Our data suggest that inhibition of platelet T×A2 production in vivo may not be picotamide’s main mechanism of action.
Immunocytochemical Study on Endothelial Markers with the Cerebral Vessel from a Patient with Familial Moyamoya Disease
Authors: Usuki Fusako , Ohtani Haruo , Okadome Toshihide , Shinmyozu Koichi , Maruyama Ikuro , Sakimoto Tetsuro , Atsuji Masayuki , Osame Mitsuhiro
Published: July 2018

The immunocytochemical study of thrombomodulin (TM), a newly recognued anticoagulant endothelial surface protein, was performed with a surgical specimen of a superficial temporalartery (STA) obtained from a 29-year-old woman with familial moyamoya disease. The staining of TM showed positive immunoreactivity in smaller vessels in the surrounding connective tissue of the specimen, whereas negative in STA. Immunoelectronmicroscopically the luminal plasma membrane of endothelial cells was positive for TM. These staining pattern was the same as that in controls. She concurrently suffered from von Willebrand disease type I, and she had two cerebral hemorrhagic attacks. A quantitative defect of the von Willebrand factor in the endothelium was demonstrated immunocytochemically.
Continuous Quantitative Monitoring of Mural, Platelet-Dependent, Thrombus Kinetics in the Crushed Rat Femoral Vein
Authors: Stockmans F , Deckmyn H , Gruwez J , Vermylen J , Acland R
Published: July 2018

A new in vivo method to study the size and dynamics of a growing mural thrombus was set up in the rat femoral vein. The method uses a standardized crush injury to induce a thrombus, and a newly developed transilluminator combined with digital analysis of video recordings. Thrombi in this model formed rapidly, reaching a maximum size 391 ± 35 sec following injury, after which they degraded with a half-life of 197 ± 31 sec. Histological examination indicated that the thrombi consisted mainly of platelets. The quantitative nature of the transillumination technique was demonstrated by simultaneous measurement of the incorporation of 111In labeled platelets into the thrombus. Thrombus formation, studied at 30 min interval in both femoral veins, showed satisfactory reproducibility overall and within a given animal

With this method we were able to induce a thrombus using a clinically relevant injury and to monitor continuously and reproducibly the kinetics of thrombus formation in a vessel of clinically and surgically relevant size
Effective Reversal of Warfarin-lnduced Excessive Anticoagulation with Low Dose Mtamin K1
Authors: Shetty H G M , Backhouse G , Bentley D P , Routledge P A
Published: July 2018

Reversal of the anticoagulant effect of warfarin in patients with no active haemorrhage can be achieved by administration of intravenous vitamin K1. Currently recommended doses of intravenous vitamin K1, for this purpose often result in subsequent difficulties in anticoagulation. We observed the response to low dose intravenous vitamin K1 in patients requiring reversal of anticoagulant therapy. Ten consecutive patients received L mg and 2l further patients received 0.5 mg of intravenous vitamin K1. In 50% of the patients who received 1 mg of vitamin K1 the INR (International Normalised Ratio) fell below 2 at 24 h whereas in patients who received 0.5 mg the INR fell below 5.5 in all subjects after 24 h and in none did it fall below 2.0. No patient had any thrombotic or haemorrhagic complications and no difficulty was encountered in re-establishing anticoagulant control after 24 h. We recommend 0.5 mg of vitamin K1 as an effective and convenient method of predictable and fine control of oral anticoagulant therapy.
Hereditary Cerebral Hemorrhage with Amyloidosis -Dutch Type: A Study of Fibrinolysis
Authors: Haan J , Kluft C , Leebeek F W G , de Bart A C W , Buruma O J S , Roos R A C
Published: July 2018

In view of reported associations between increased bleeding tendency and systemically decreased α2-antiplasmin in patients with systemic amyloid deposition we studied α2-antiplasmin, fibrinogen, C-reactive protein and blood levels of locally produced endothelial hemostasis factors in the acute and quiescent phase in 16 patients with hereditary cerebral hemorrhage with amyloidosis - Dutch type (HCHWA-D).

None of the factors measured in the quiescent phase of the disease was abnormal. In the acute phase, shortly after a stroke, only factor VIII: Ag was evidently elevated. We concluded that systemic abnormalities in the part of the fibrinolysis system studied are not likely to be responsible for multifocal and recurrent cerebral hemorrhages in HCHWA-D. The role of an elevated factor VIII: Ag level in the acute phase is unclear.
The Mode of Action of CY216 and CY222 in Plasma
Authors: Béguin Suzette , Wielders Simone , Lormeau J C , Hemker H Coenraad
Published: July 2018

Three fractions of the low molecular weight heparin CY216 (fraxiparin, mean molecular weight [MMW] 5,090), with MMWs of respectively, 3,090, 4,400 and 7,910 were prepared by gel permeation chromatography. From CY222 (MMW 3,770) as well as from CY216 and its three fractions the material with high affinity to antithrombin III (AT III) was obtained by chromatography on immobilised AT III. The molecular weight distribution of each of the ten preparations thus obtained was determined by high performance liquid chromatography, while the content of AT III binding material was determined by stoichiometric titration of AT III, monitored by intrinsic fluorescence enhancement.

We measured the effect of all heparins on the decay of endogenous thrombin in plasma and on the overall generation of thrombin in plasma, triggered via the extrinsic or via the intrinsic pathway. From these data we calculated the time course of prothrombin conversion, i. e. the course of factor Xa activity as expressed by prothrombinase activity.

It was found that in platelet-poor plasma the anticoagulant properties of the heparins are largely dependent on their antithrombin action, which is determined by their content of high affinity material with a MW of 5,400 or higher. The specific antithrombin activity of all heparins, when expressed in terms of material with high affinity to antithrombin III (HAM) with a MW >5,400 is 13.0 min-1/(μg/ml) (range 10.5-15.9). The anticoagulant potency is not influenced by the presence of low-affinity material and hardly by material with MW <5,400.

In platelet-rich plasma, however, the presence of non-AT III binding material enhances the inhibition, presumably by neutralising heparin binding material originating from activated platelets. The ultra low MW fractions (<3,400) show a similar activity in PPP and in PRP.
Characterization of Human Tissue-Type Plasminogen Activator with Monoclonal Antibodies: Mapping of Epitopes and Binding Sites for Fibrin and Lysine
Authors: Zacharias Ute , Fischer Bernhard , Noll Franz , Will Horst
Published: July 2018

The study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding.

Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D’). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA.

The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F. Weak inhibition is exerted on association of mAb 2, 21, and 25 to epitope D. Amidolytic activity is suppressed only by B-chain specific antibody 22.
Transport of Anti-Glycoprotein IIb/IIIa-Antibodies into the Alpha-Granules of Unstimulated Human Blood Platelets
Authors: Morgenstern Eberhard , Ruf Andreas , Patscheke Heinrich
Published: July 2018

The redistribution of the antibody-glycoprotein (GP) IIb/IIIa complex was investigated with the immuno-gold labeling technique in order to trace its transport in resting platelets. Washed platelets were incubated in the presence of aspirin and a prostacyclin analogue (iloprost) with three different monoclonal antibodies (Gi3, J15 and P2) against GPIIb/IIIa. The examination of ultrathin serial sections showed that the surface labeling was internalized into the surface connected membrane system (SCS). Labels were found within the alpha-granules after 40 min and the number of labels increased during longer incubation periods (max. 120 min). The transport possibly involved coated membranes. The alpha-granules were neither found to be altered during this process nor were any morphological signs of platelet activation detectable. The anti-GPIIb/IIIa complex remained membrane-associated during the transfer. These observations indicate that the membrane-GPIIb/IIIa complex was stable and transported from the surface into the alpha-granules of resting platelets. Since this transport was not influenced by iloprost or by aspirin it may be interpreted as constitutive endocytosis.
Effect of Calcium Channel Blockers on Platelet GPIIb-IIIa as a Calcium Channel in Liposomes: Comparison with Effects on the Intact Platelet
Authors: Rybak Mary Ellen M , Renzulli Lori A
Published: July 2018

The platelet membrane glycoprotein IIb-IIIa complex is essential for platelet aggregation and functions as a fibrinogen receptor on the activated platelet. When incorporated into phospholipid vesicles, this glycoprotein complex can function as an apparent calcium channel which facilitates the transit of calcium across a phospholipid barrier. In order to further evaluate this calcium channel, the effect of calcium channel blockers of the dihydropyridine (nifedipine and nicardipine), arylalkylamine (verapamil) and benzothiazipine (diltiazem) classes were evaluated on GPIIb-Illa liposomes with encapsulated fura-2 (a fluorescent calcium indicator). Nicardipine, verapamil, and nifedipine significantly inhibited calcium influx into GPIIb-IIIa liposomes; however, this required 190 μM, 400 μM, and 140 μM drug, respectively. These concentrations are 10-1,000 fold greater than those clinically obtainable. In contrast, diltiazem at concentrations >220 μM and amiloride at concentrations >800 μM showed no inhibitory effects. When aspirinized platelets were activated with 30 μg/ml bovine fibrillar collagen, both nicardipine and diltiazem produced a decrease in both the initial rise and maximum cytoplasmic calcium concentration. Parallel experiments were performed to assess the effects of verapamil, nicardipine, and diltiazem on platelet aggregation in platelet rich plasma. Nicardipine, 190-380 μM, induced a prolongation of the lag phase, but no effect on the final degree of platelet aggregation to collagen. Similar inhibition of platelet aggregation was seen with diltiazem and verapamil although the effect of diltiazem was less pronounced particularly at higher concentrations of collagen. No effect was seen on aggregation with 32 μM ADP which is release independent, or on the primary wave of low dose ADP induced platelet aggregation. Verapamil (200 μM) and nicardipine (>190 μM) partially inhibited the secondary wave of 3.2 μM ADP aggregation. In contrast, diltiazem (400 μM) had limited effect on ADP induced aggregation. Similarly, both nicardipine and verapamil markedly inhibited platelet aggregation to 6 μM epinephrine; however, diltiazem had no effect. These data demonstrate that the GPIIb-IIIa calcium channel is selectively inhibited by different classes of calcium channel blockers. This inhibition requires concentrations much higher than that to inhibit classic voltage gated calcium channels and may be by a different mechanism. Further evaluation will determine whether this differential inhibition of the GPIIb-IIIa calcium channel is related to differences observed in platelet aggregation.
Neutralization of the Antiheparin Activity of Platelet Factor 4 by a Monoclonal Antibody
Authors: Saggin Leopoldo , Cazzola Flavia , Corona Giuseppe , Salvatico Emanuela , Cella Giuseppe , Prosdocimi Marco
Published: July 2018

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4’s ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/ PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.
Fine Mapping of Monoclonal Antibody Epitopes on Human von Willebrand Factor Using a Recombinant Peptide Library
Authors: Ginsburg David , Bockenstedt Paula L , Allen Elizabeth A , Fox David A , Foster Paul A , Ruggeri Zaverio M , Zimmerman Theodore S , Montgomery Robert R , Bahou Wadie F , Johnson Timothy A , Yang Angela Y
Published: July 2018

A recombinant human von Willebrand factor (vWF) cDNA fragment library was constructed in λgtll for the localization of anti-vWF monoclonal antibody epitopes. Twelve of 21 monoclonal antibodies screened identified epitopes expressed in λgtll as β-galactosidase fusion proteins. By sequence analysis, these antigenic determinants were localized to segments ranging from 17 to 105 amino acids in length. Four epitopes apparently shared by more than one antibody were identified, suggesting the presence of immuno-dominant epitopes within vWF. Monoclonal antibody C3, which blocks factor VIII (FVIII) binding to vWF, bound to the same epitope previously identified by a second monoclonal antibody which also blocks this function, suggesting that this region may be at or near the vWF/FVIII binding domain. Three antibodies recognize the same region within the vWF A2 repeat. Mutations near this region appear to be responsible for Type IIA von Willebrand’s disease. The co-localization of these antibodies suggests that this domain might be exposed on the surface of vWF, consistent with its apparent increased sensitivity to plasma proteases.
Partial Characterization of an Autoantibody Recognizing the Secondary Binding Site(s) of Thrombin in a Patient with Recurrent Spontaneous Arterial Thrombosis
Authors: Costa J M , Fiessinger J N , Capron L , Aiach M
Published: July 2018

A patient with an 18 year history of recurrent arterial thrombosis and no evidence of atherosclerosis or embolism of cardiac origin presented with a prolonged thrombin clotting time when performed with human thrombin. The bovine thrombin clotting time was only slightly prolonged. During 30 months of follow-up, the thrombin time fluctuated, but remained prolonged. The patient has been treated with an oral anticoagulant for the past 8 years, with no thrombotic recurrence.

The inhibitor activity was due to the presence of polyclonal IgGs which bound to thrombin-Sepharose. The influence of IgGs purified from the patient’s serum was compared to the influence of normal IgGs in several systems exploring the catalytic activity of thrombin and the binding of the enzyme to macromolecular substrates through secondary binding site(s). We found that the IgGs did not impair the catalytic activity toward small synthetic substrates, but inhibited the binding of thrombin to fibrinogen, thrombomodulin and heparin cofactor II. Such proteins are known to require a secondary binding site of thrombin to interact with the enzyme.

The anti-thrombin antibody might have resulted from an abnormal generation of thrombin. This would be the consequence of the process favouring thrombosis. Alternatively, the autoantibody might have favoured thrombosis primarily, by impairing natural antithrombotic mechanisms triggered by thrombin.
Markers of Procoagulant Imbalance in Patients with Inherited Thrombophilic Syndromes
Authors: Mannucci P M , Tripodi A , Bottasso B , Baudo F , Finazzi G , Stefano V De , Palareti G , Manotti C , Mazzucconi M G , Castaman G
Published: July 2018

In 107 asymptomatic and untreated patients with inherited syndromes associated with thrombophilia (antithrombin III, protein C and protein S deficiencies), we compared in parallel two plasma peptides which reflect activation of the common coagulation pathway: the prothrombin fragment 1 + 2 (F1+2) and fibrinopeptide A (FPA). Both F1+2 and FPA were measured with simple, commercially available ELISA methods. High levels of F1+2 or FPA were found in about one fourth of the patients as a whole. When patients were divided according to the type of inherited thrombophilie syndrome, it appeared that F1+2 was more frequently elevated in protein C and protein S deficiencies than in antithrombin deficiency; and that, in general, it was no more frequently elevated than FPA. Although our data confirm the existence of a procoagulant imbalance in inherited thrombophilie syndromes due to defects of natural anticoagulant proteins, they do not confirm that such imbalance can be more frequently diagnosed by measuring F1+2 levels, particularly in patients with antithrombin deficiency.
Thrombomodulin Is a Cofactor for Thrombin Degradation of Recombinant Single-Chain Urokinase Plasminogen Activator "In Vitro" and in a Perfused Rabbit Heart Model
Authors: Molinari Antonio , Giorgetti Carla , Lansen Jacqueline , Vaghi Fabrizio , Orsini Gaetano , Faioni Elena Maria , Mannucci Pier Mannuccio
Published: July 2018

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolyti-cally inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect.

We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.
Determinants of Induction of Increased Synthesis of Plasminogen Activator Inhibitor Type-1 in Human Endothelial Cells by t-PA
Authors: Fujii Satoshi , Sobel Burton E
Published: July 2018

Tissue-type plasminogen activator (t-PA) induces synthesis of a rapidly acting inhibitor, plasminogen activator inhibitor type-1 (PAI-1) in human umbilical vein endothelial cells (HUVEC) in culture. In vivo, an analogous process may induce negative feedback on the fibrinolytic system. To define specific determinants in the t-PA molecule contributing to the induction, PAI-1 synthesis was characterized in 35S-methionine labeled HUVEC in response to several molecular variants of t-PA. Catalytically active variants devoid of several specific structural domains in the A-chain retained the capacity to form complexes with PAI-1 and to induce increased concentrations of total PAI-1 (free and complexed) in conditioned media without depleting PAI-1 from the extracellular matrix. Surprisingly, a mutant t-PA with markedly reduced catalytic activity reflecting replacement of the active site serine with threonine (S478T) formed complexes with PAI-1 and induced increased PAI-1 synthesis as well. However, in contrast to wild-type t-PA and A-chain variants, it did not release 35S-methionine labeled PAI-1 from the extracellular matrix. Thus, its effects appeared to reflect increased secretion exclusively. Our results suggest that induction of PAI-1 synthesis in HUVEC by t-PA depends on its protease domain but that an active site serine is not a requirement.
Alternative Splicing Is Responsible for the Presence of Two Tissue Factor mRNA Species in LPS Stimulated Human Monocytes
Authors: Paul C , van der Logt E , Reitsma Pieter H , Bertina Rogier M
Published: July 2018

Although normally absent from the surface of all circulating cell types, tissue factor (TF) can be induced to appear on circulating monocytes by stimulants like bacterial lipopolysaccharide (LPS) and phorbolesters. Northern analysis of RNA isolated from LPS stimulated human monocytes demonstrates the presence of 2.2 kb and 3.1 kb TF mRNA species. The 2.2 kb message codes for the TF protein. As demonstrated by Northern blot analysis with a variety of TF gene probes, the 3.1 kb message arises from an alternative splicing process which fails to remove 955 bp from intron 1. Because of a stop codon in intron 1 no TF protein is produced from the 3.1 kb transcript. This larger transcript should therefore not be taken into account when comparing TF gene transcription and TF protein levels.