bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence on Platelet Function by Heparin in Men with Unstable Coronary Artery Disease
Authors: Berglund Ulf , Wallentin Lars
Published: July 2018

Ninetyseven men with unstable coronary artery disease (CAD), i.e. unstable angina or a non Q-wave myocardial infarction, entered a double-blind placebo-controlled study with heparin intravenously 30,000-40,000 U daily. Platelet function was evaluated as ex vivo aggregation toward collagen and ADP and as the platelet inhibitory effect of prostacyclin, before and after 5 days of treatment. Heparin increased aggregation induced by ADP 1 εM from 39.6 ± 3.1% to 52.1 ± 4.1% (p = 0.014) and reduced the inhibition of aggregation by prostacyclin 1.0 ng/ml from 59.6 ± 3.7% to 39.3 ± 5.6% (p <0.001). No changes occurred in the placebo group.

Thus, treatment with heparin enhances the platelet sensitivity to ADP and decreases the platelet inhibitory effect of prostacyclin in men with unstable CAD. Concomitant treatment with acetylsalicylic acid abolishes the increased reponse to ADP, but does not seem to influence the interaction between heparin and prostacyclin.
Antithrombin III: A Database of Mutations
Authors: Lane D A , Ireland H , Olds R J , Thein S L , Perry D J , Aiach M
Published: July 2018

Elucidation of the molecular defects reponsible for antithrombin III deficiency is proceeding rapidly. In order that a record is kept of the new and duplicated mutations that are found, we have compiled a database that we plan to update annually. In this, the first report of the database, we list 6 antithrombin III locus sequence polymorphisms and 94 recorded mutations causing functional deficiency of the protein, 38 of which are novel. As is the case with mutations affecting other protein genes, most mutations of antithrombin III involve a CG to TG or CA change.
Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate
Authors: Cattaneo Marco , Akkawat Benjaporn , Lecchi Anna , Cimminiello Claudio , Capitanio Anna M , Mannucci Pier Mannuccio
Published: July 2018

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrihogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPIIb/IIIa monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their K
d were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.
Comparison of Factor VIII Levels after Adrenalin in Classic Hemophilia and von Willebrand’s Disease (vWd)
Authors: Eyster M Elaine , Ballard James O , Prager David
Published: July 2018

The factor VIII response to adrenalin was compared in 5 patients with von Willebrand’s disease (vWd) and 10 hemophiliacs with baseline factor VIII procoagulant activity (VIIIAHF) of 0.01 u/ml or greater. Patients with vWd showed a two-fold VIII AHF increase, while those with hemophilia showed no significant change 1 hr following the subcutaneous injection of adrenalin. These results suggest that the adrenalin stimulation test may be of value in distinguishing certain patients with mild hemophilia from those with vWd.
Platelet Adhesion to Collagen in Subtypes of Type I von Willebrand’s Disease Is Dependent on Platelet von Willebrand Factor
Authors: d'Alessio Patrizia , Zwaginga Jaap Jan , de Boer Hetty C , Federici Augusto B , Rodeghierio Francesco , Castaman Giancarlo , Mariani Guglielmo , Msnnucci P Mannuccio , de Groot Phillip G , Sixma Jan J
Published: July 2018

Von Willebrand’s disease type I, characterized by low levels of factor VIII coagulant activity (VTII :C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (RiCof) (1), can be subdivided on the basis of platelet von Willebrand factor into subtype platelet normal, platelet discordant, and platelet low (2). We have investigated the contribution of platelet von Willebrand factor in these various subtypes to platelet adhesion using the rectangular perfusion chamber of Sakariassen et al. (3) with fibrillar collagen or a fibroblast matrix as adhesive surfaces. Platelet adhesion to fibrillar collagen was decreased in all subtypes of von Willebrand’s disease, but not as low as in severe von Willebrand’s disease. A close correlation was observed between platelet adhesion to collagen and plasma vWF:Ag in severe von Willebrand’s disease, subtype platelet low, subtype platelet discordant, and normal controls. The platelet adhesion in subtype platelet normal was higher than expected from the plasma vWF: Ag level. Perfusions in which washed platelets were added to a human albumin solution together with red blood cells gave similar adhesion values in subtype platelet normal and normal controls; adhesion was decreased in subtype platelet discordant, and the lowest values were found in subtype platelet low and in severe von Willebrand’s disease. These data indicate that platelet von Willebrand factor may contribute to platelet adhesion, when plasma von Willebrand factor is low. Perfusion studies over a fibroblast matrix gave similar low adhesion values for subtype platelet low and platelet normal, indicating that the contribution of platelet von Willebrand factor can only be observed on a strongly activating surface such as fibrillar collagen.
Purification and Properties of Prothrombin Modified by Asbestos Filtration of Human Plasma
Authors: Izuchi Y , Boffa M C , Soulier J P
Published: July 2018

Filtration through asbestos filter (Seitz) of human plasma modified the prothrombin molecule as previously shown. Factor II could no longer be activated by physiological activators (Ca++ + phospholipid + V and Xa) but reacted readily with staphylocoagulase. The separation and purification of the modified prothrombin allowed the preparation of two fractions:

A small slightly modified accessory fraction, “prothrombin-asbestos- 1”, lost its ability to be activated by physiological activators, and its ability to be adsorbed by barium citrate, but retained the immunological reactivity of fragment 1, as well as the mobility and molecular weight of unmodified prothrombin.

A main fraction, “prothrombin-asbestos-2” appeared to be a modified prothrombin which has lost its N-terminal extremity. It was not adsorbed by barium citrate and could not be activated by physiological activators. It possessed a reduced electrophoretic mobility, as well as a reduced molecular weight (39,000), which are properties similar to those of thrombin. Both fractions 1 and 2 were devoid of thrombin activity.

Asbestos was thus able to break the prothrombin molecule non enzymatically, the amputation of the N terminal extremity being responsible for the new functional and physicochemical properties of the molecule.

Staphylocoagulase appeared not to need the N terminal extremity of the molecule of prothrombin to form the active thrombin-coagulase complex.
Haemostatic Factors and Inhibitors and Coronary Artery Bypass Grafting: Preoperative Alterations and Relations to Graft Occlusion
Authors: Moor Elisabeth , Hamsten Anders , Blombäck Margareta , Herzfeld Istvan , Wiman Björn , Rydén Lars
Published: July 2018

Graft closure remains a major problem after coronary artery bypass surgery. While a number of graft characteristics influencing the risk of occlusion have been defined, the role of haemostatic factors and inhibitors has not been studied in detail. The present study examined the time course of changes in blood coagulation and fibrinolytic function after coronary artery bypass grafting in 20 consecutive patients. Pre- and postoperative determinations of haemostatic factors and inhibitors were also related to the presence of graft occlusion assessed by angiography at three months after surgery. A broad panel of haemostatic tests was used preoperatively, on the first, third and eight postoperative days, and at three months after surgery. A particular emphasis was placed on fibrinogen, factor VII activity, von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1) activity, anticoagulant proteins C and S, thrombin-antithrombin complex and D-dimer. A marked activation of the coagulation cascade was noted postoperatively along with enhanced degradation of cross-linked fibrin. The degree of activation of blood coagulation and fibrinolysis differed widely between individuals and appeared to relate only partly to the acute phase reaction produced by the surgical trauma. Preoperative values of haemostatic factors and inhibitors showed fairly weak associations with the levels of postoperative determinations. Basal tPA and factor VIII levels, fibrinogen and TAT concentrations on the third and eighth postoperative day, and factor VII amidolytic activity on the third postoperative day differed (p <0.05) between patients with and without occluded grafts at reangiography. Accordingly, combined pre- and postoperative assessment of haemostatic function may contribute to the identification of individuals at risk for early graft closure after coronary artery bypass grafting.
Neutrophil but not Monocyte Activation Inhibits P-Selectin-Mediated Platelet Adhesion
Authors: Rinder Henry M , Tracey Jayne L , Rinder Christine S , Leitenberg David , Smith Brian R
Published: July 2018

Selectins are Ca2+-dependent glycoprotein receptors that mediate the adhesion of activated platelets or endothelial cells to unstimulated leukocytes. Using purified cell fractions, we examined activated neutrophil adhesion to P-selectin-expressing platelets and found that phorbol 12-myristate 13-acetate (PMA), platelet activating factor C16 (PAF), and n-formyl-met-leu-phe (fMLP) pretreatment of neutrophils inhibited activated platelet adhesion. Furthermore, PMA and PAF were capable of dissociating established resting neutrophil-activated platelet conjugates. Since L-selectin is downregulated after leukocyte activation and has been postulated as a ligand for P-selectin, we preincubated resting neutrophils with Dreg-2 and Dreg-56, blocking monoclonal antibodies (MoAb) to L-selectin; these MoAb failed to inhibit activated platelet adhesion. To more closely approximate in vivo conditions of leukocyte and platelet activation, we also employed a whole blood (WB) model of leukocyte-platelet adhesion. We found that simultaneous activation of both platelets and leukocytes by PMA caused an immediate rise in the % of P-selectin-positive platelets accompanied by a rapid increase in monocyte-platelet and neutrophil-platelet conjugates; however, the % of neutrophil-platelet conjugates subsequently declined over 30-60 min to baseline levels while monocyte-platelet adhesion remained elevated over 90 min. By contrast, selective platelet activation in WB by thrombin resulted in an increase in platelet P-selectin expression accompanied by a sustained (90 min) elevation in both monocyte- and neutrophil-platelet conjugates. This increase in leukocyte-platelet conjugates after thrombin was not inhibited by preincubation of WB with Dreg-2 or Dreg-56. We conclude that neutrophil activation decreases the expression of the ligand for platelet P-selectin within 30-60 min resulting in inhibition of neutrophil-platelet adhesion and dissociation of existing neutrophil-platelet conjugates. By contrast, monocyte activation over 90 min does not affect monocyte adhesion to activated platelets in whole blood.
Is Colour Doppler Ultrasound a Sensitive Screening Method in Diagnosing Deep Vein Thrombosis after Hip Surgery ?
Authors: Magnusson Marie , Eriksson Bengt I , Kãlebo Peter , Sivertsson Ramon
Published: July 2018

Patients undergoing orthopedic surgery are at high risk of developing deep vein thrombosis. One hundred and thirty-eight consecutive patients undergoing total hip replacement or hip fracture surgery were included in this study. They were surveilled with colour Doppler ultrasound (CDU) and bilateral ascending contrast phlebography. The prevalence of proximal and distal DVT in this study was 5.8% and 20.3% respectively.

CDU has a satisfactory sensitivity in patients with symptomatic deep vein thrombosis, especially in the proximal region. These results could not be confirmed in the present study of asymptomatic patients. The sensitivity was 62.5% (95% confidence interval: C.I. 24-91%) and the specificity 99.6% (C.I. 98-100%) for proximal DVT; 53.6% (C.I. 34-73%) and 98% (C.I. 96-99%) respectively for distal thrombi. The overall sensitivity was 58.1% (C.I. 39-75%) and the specificity 98% (C.I. 96-99%). The positive predictive value was 83.3% (C.I. 36-99%) and 75% (C.I. 51-91%) for proximal and distal DVT respectively. The negative predictive value was 98.9% (C.I. 98-100%) and 94.9% (C.I. 92-98%) for proximal and distal DVT respectively. The results of this study showed that even with a highly specialised and experienced investigator the sensitivity of CDU was too low to make it suitable for screening purposes in a high risk surgical population.
The Gla26 Residue of Protein C Is Required for the Binding of Protein C to Thrombomodulin and Endothelial Cell Protein C Receptor, but not to Protein S and Factor Va
Authors: Nishioka Junji , Ido Masaru , Hayashi Tatsuya , Suzuki Koji
Published: July 2018

A functionally defective protein C (PC)-Mie, detected in the plasma of a patient with hereditary thrombophilia, has Lys substituted for γ-carboxyglutamic acid (Gla)26 residue. The activation rate of PC-Mie by Protac or thrombin in the absence of Ca2+ and that by thrombin with native thrombomodulin (TM), recombinant soluble truncated TM or on cultured endothelial cells in the presence of Ca2+ were all apparently lower than that of normal PC. The anticoagulant activity of Protac-activated PC (APC)-Mie on the plasma clotting time and the rate of inactivation of factor Va by APC-Mie in the presence of phospholipids were lower than those seen with normal APC. APC-Mie and normal APC bound equally to protein S and to biotinyl-factor Va. However, neither PC-Mie nor APC-Mie bound to phospholipids and to cultured human endothelial cells. It was similar to that observed with Gla-domainless PC/APC, but different from that seen with normal PC/APC. These results suggest that Gla26-dependent conformation is required for the binding of PC/APC to phospholipids, TM and the surface of endothelial cell PC/APC receptor, but not to protein S and factor Va.
Low Molecular Weight Heparin Is Responsible for the Anti-Xa Activity of Desmin 370
Authors: Brieger David , Dawes Joan
Published: July 2018

Dermatan sulphate does not catalyse the inactivation of factor Xa. However, the low molecular weight (LMW) dermatan sulphate Desmin 370 has been shown to generate circulating anti-Xa activity following administration to humans. Using a single batch of Desmin 370, we measured 3 U/mg of anti-Xa activity by amidolytic assay in vitro. The material responsible for this activity had a lower molecular weight range (6000 and 1800 Da) than Desmin 370 and was more highly sulphated than the bulk of the drug. Heparinase digestion of Desmin 370 eliminated 90% of the in vitro anti-Xa activity without significantly interfering with its ability to potentiate inactivation of thrombin by HCII, suggesting that the anti-Xa activity is not due to dermatan sulphate and is probably heparin. When 125I-labelled Desmin 370 together with 40 mg/kg carrier drug was administered intravenously to a rabbit, anti-Xa activity was readily detectable in the plasma for up to 10 h and had a longer half-life than the sulphated radiolabel. Most of this anticoagulant activity was recovered from the plasma by Polybrene affinity chromatography and was probably a sulphated glycosaminoglycan. Administration of the heparinase-digested drug to a rabbit resulted in 70% less anti-Xa activity than the undigested drug. We conclude that Desmin 370 contains detectable quantities of biologically active low molecular weight heparin, which is responsible for persistent anti-Xa activity following intravenous administration.
Echinomycin Suppresses the Pyrogenic Effects of Endotoxin and Interleukin- lβ in Human Endothelial Cells and Peripheral Blood Mononuclear Cells
Authors: Herbert Jean-Marc , Lalé Alain , Pereillo Jean-Marie , Derocq Jean-Marie , Casellas Pierre
Published: July 2018

Endotoxin (LPS) and interleukin-1(3 (IL-iβ) increased in a dose-dependent manner the expression of tissue factor, an ubiquitous membrane-anchored glycoprotein that initiates blood coagulation at the surface of human umbilical vein endothelial cells (HUVEC) and human peripheral blood mononuclear cells (PBMC). Echinomycin, a cyclic octapeptide of microbial origin strongly inhibited LPS- and IL-1β-induced tissue factor expression in HUVEC and PBMC with IC50 values in the subnanomolar range at the same time it reduced LPS and IL-iβ-induced expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on HUVEC (IC50 = 0.4 ± 0.1 and 0.3 ± 0.2 nM respectively). Echinomycin also reduced LPS-induced secretion of IL-iβ and IL-6 by human PBMC (IC50 = 10 ± 2 and 3 ± 0.5 nM respectively). These observations demonstrate that echinomycin protects endothelial cells and PBMC from the pyrogenic effect of LPS and 1β.
Thrombosis in Cancer Patients Treated with Hematopoietic Growth Factors - A Meta-Analysis
Authors: Barbul T , Finazzi G , Grassi A , Marchioli R
Published: July 2018

Hematopoietic colony-stimulating factors (CSFs) are largely used in patients with cancer undergoing cytotoxic treatment to accelerate neutrophil recovery and decrease the incidence of febrile neutropenia. Clinical practice guidelines for their use have been recently established (1), taking into account clinical benefit, but also cost and toxicity. Vascular occlusions have been recently reported among the severe reactions associated with the use of CSFs, in anedoctal case reports (2, 3), consecutive case series (4) and randomized clinical trial (5, 6). However, the role of CSFs in the pathogenesis of thrombotic complications is difficult to ascertain, because pertinent data are scanty and widely distributed over a number of heterogenous investigations. We report here a systematic review of relevant articles, with the aims to estimate the prevalence of thrombosis associated with the use of CSFs and to assess if this rate is significantly higher than that observed in cancer patients not receiving CSFs.
Tissue Factor Induces Migration of Cultured Aortic Smooth Muscle Cells
Authors: Sato Yuichiro , Asada Yujiro , Marutsuka Kousuke , Hatakeyama Kinta , Sumiyoshi Akinobu
Published: July 2018

Tissue factor (TF) plays a key role as a primary initiator on the extrinsic coagulation cascade. Recently, upregulation of TF has been reported in human atherosclerotic lesions. We investigated the effects of TF on migration and proliferation of cultured smooth muscle cells (SMCs) from rabbit aortas. We tested three kinds of recombinant human TF (L-TF: the full length of TF with relipidation, NL-TF: the full length of TF without relipidation, and S-TF: a soluble form of TF1-219). Only L-TF had coagulant activity. All kinds of TF showed the chemotactic migration activity for SMCs. The migration ability of TFs was comparable to those of platelet-derived growth factor (PDGF)-BB and basic fibroblast-growth factor (bFGF), and was inhibited by anti-TF polyclonal and monoclonal antibodies. On the other hand, none of the forms of TF induced SMC proliferation. These results indicate that TF is not only a coagulation factor but also a strong chemotactic factor for vascular SMCs, and suggest that TF could play an important role in atherogenesis as well as in hemostasis and thrombosis.
“Pseudo Homozygous” Activated Protein C Resistance due to Double Heterozygous Factor V Defects (Factor V Leiden Mutation and Type I Quantitative Factor V Defect) Associated with Thrombosis: Report of Two Cases Belonging to Two Unrelated Kindreds
Authors: Simioni Paolo , Scudeller Alberta , Radossi Paolo , Gavasso Sabrina , Girolami Bruno , Tormene Daniela , Girolami Antonio
Published: July 2018

Two unrelated patients belonging to two Italian kindreds with a history of thrombotic manifestations were found to have a double heterozygous defect of factor V (F. V), namely type I quantitative F. V defect and F. V Leiden mutation. Although DNA analysis confirmed the presence of a heterozygous F. V Leiden mutation, the measurement of the responsiveness of patients plasma to addition of activated protein C (APC) gave results similar to those found in homozygous defects. It has been recently reported in a preliminary form that the coinheritance of heterozygous F. V Leiden mutation and type I quantitative F. V deficiency in three individuals belonging to the same family resulted in the so-called pseudo homozygous APC resistance with APC sensitivity ratio (APC-SR) typical of homozygous F. V Leiden mutation. In this study we report two new cases of pseudo homozygous APC resistance. Both patients experienced thrombotic manifestations. It is likely that the absence of normal F. V, instead of protecting from thrombotic risk due to heterozygous F. V Leiden mutation, increased the predisposition to thrombosis since the patients became, in fact, pseudo-homozygotes for APC resistance. DNA-analysis is the only way to genotype a patient and is strongly recommended to confirm a diagnosis of homozygous F. V Leiden mutation also in patients with the lowest values of APC-SR. It is to be hoped that no patient gets a diagnosis of homozygous F. V Leiden mutation based on the APC-resi-stance test, especially when the basal clotting tests, i.e., PT and aPTT; are borderline or slightly prolonged.
Antibodies to Prothrombin Imply a Risk of Myocardial Infarction in Middle-Aged Men
Authors: Vaarala Outi , Puurunen Marja , Mänttäri Matti , Manninen Vesa , Aho Kimmo , Palosuo Timo
Published: July 2018

Antiphospholipid antibodies in patients with “antiphospholipid syndrome” may be directed at least in part against plasma phospholipid-binding proteins, such as Β2-glycoprotein I or prothrombin, which are involved in the control of thrombosis and haemostasis. IgG-class antibodies against prothrombin and Β2-glycoprotein I were measured by enzyme-linked immunoassay in initially healthy middle-aged dyslipid-aemic men (non-high-density lipoprotein >5.2 mml/1). Serum samples had been drawn at entry to a 5-year coronary primary-prevention trial with gemfibrozil from 106 subjects who experienced either a non-fatal myocardial infarction or cardiac death during the follow-up and from 106 subjects without coronary episodes, matched for treatment group (gemfibrozil/placebo) and geographical area.

The antiprothrombin antibody level, as expressed in optical density units, was significantly higher in patients than in controls (0.26 ±0.17 versus 0.22 ±0.09; p<0.02). A high level of antiprothrombin antibodies (highest tertile of distribution) predicted a 2.5-fold increase in the risk (95% confidence interval 1.2-5.3) of myocardial infarction or cardiac death. The distribution of IgG-class antibodies against (Β2-glycoprotein I did not differ significantly between cases and controls. The joint effect of antiprothrombin antibodies and other factors associated with hypercoagulative state: triglyceride level, lipoprotein(a) and smoking, was multiplicative for the risk. Antiprothrombin antibodies are a new immunological predictor of myocardial infarction and the effect of these antibodies may be mediated by hypercoagulative mechanisms.
Interventional Thermal Injury of the Arterial Wall: Unfolding of von Willebrand Factor and Its Increased Binding to Collagen after 55° C Heating
Authors: Post Mark J , de Graaf-Bos Anke N , Posthuma George , de Groot Philip G , Sixma Jan J , Borst Cornelius
Published: July 2018

Purpose. Thermal angioplasty alters the thrombogenicity of the arterial wall. In previous studies, platelet adhesion was found to increase after heating human subendothelium to 55° C and decrease after heating to 90° C. In the present electron microscopic study, the mechanism of this temperature-dependent platelet adhesion to the heated arterial wall is elucidated by investigating temperature-dependent conformational changes of von Willebrand factor (vWF) and collagen types I and III and the binding of vWF to heated collagen.

Methods. Purified vWF and/or collagen was applied to electron microscopic grids and heated by floating on a salt-solution of 37° C, 55° C or 90° C for 15 s. After incubation with a polyclonal antibody against vWF and incubation with protein A/gold, the grids were examined by electron microscopy.

Results. At 37° C, vWF was coiled. At 55° C, vWF unfolded, whereas heating at 90° C caused a reduction in antigenicity. Collagen fibers heated to 37° C were 60.3 ± 3.1 nm wide. Heating to 55° C resulted in the unwinding of the fibers, increasing the width to 87.5 ± 8.2 nm (p < 0.01). Heating to 90° C resulted in denatured fibers with an enlarged width of 85.1 ± 6.1 nm (p < 0.05). Heating of collagen to 55° C resulted in an increased vWF binding as compared to collagen heated to 37° C or to 90° C. Incubation of collagen with vWF, prior to heating, resulted in a vWF binding after heating to 55° C that was similar to the 37° C binding and a decreased binding after 90° C.

Conclusions. After 55° C heating, the von Willebrand factor molecule unfolds and collagen types I and III exhibit an increased adhesiveness for von Willebrand factor. Heating to 90° C denatures von Willebrand factor and collagen. The conformation changes of von Willebrand factor and its altered binding to collagen type I and III may explain the increased and decreased platelet adhesion to subendothelium after 55° C and 90° C heating, respectively.
Species Specificity of Anti-β2 Glycoprotein I Autoantibodies and Its Relevance to Anticardiolipin Antibody Quantitation
Authors: Arvieux J , Darnige L , Hachulla E , Roussel B , Bensa J C , Colomb M G
Published: July 2018

Some patients suspected of having antiphospholipid antibody syndrome (APS) were found to be positive for anti-β2 glycoprotein I (β2GPI) antibodies despite negative results for antibodies to cardiolipin (ACA). Since the major source of β2GPI in the ACA assay is animal (usually bovine) serum, we studied the influence on ACA quantitation of the species specificity of anti-β2GPI antibodies from patients with various autoimmune disorders, mostly systemic lupus erythematosus and primary APS. Ninety-seven sera were selected based on IgG (n = 76) or IgM (n = 64) positivity by ELISA using γ-irradiated plates coated with human or bovine purified β2GPI. A higher proportion of IgM (43.7%) than IgG (7.9%) reacted to human, but not bovine, β2GPI. Furthermore, from the samples reactive to both proteins, the ratio of antibody level against bovine to that against human β2GPI was 1.08 ± 0.58 for IgG and 0.58 ± 0.3 for IgM (p <10−5). IgG and IgM ACA were detected in 78 and 40 sera, respectively; concordance between the two ELISAs for ACA and anti-β2GPI antibodies was 94% for IgG and 75% for IgM. Out of 28 IgM showing recognition restricted to human β2GPI, 21 were missed by the ACA assay, possibly because of lower concentrations of β2GPI in those patients’ sera. The antibody reactivity pattern towards human and bovine β2GPI of individual sera showed no variation with time and was related to the relative antibody avidity for each protein. A murine anti-human β2GPI monoclonal antibody, 9G1, that cross-reacts with bovine β2GPI, competed to a large extent with the patients’ anti-β2GPI antibody binding sites whatever isotype involved or protein recognized. Therefore, anti-β2GPI antibodies of IgM isotype display a marked preference for human compared to bovine β2GPI responsible for frequent inconsistencies in the ACA assay.
Evaluation of a Low Molecular Weight Modulator of Human Plasminogen Activator Inhibitor-1 Activity
Authors: Charlton Peter A , Faint Richard W , Bent Fiona , Bryans Justin , Chicarelli-Robinson Inês , Mackie Ian , Machin Samuel , Bevan Paul
Published: July 2018

A critical component in the regulation of thrombus formation and clearance is the balance between tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAM). An increase in the plasma concentration of PAH has been proposed as a risk factor in thrombotic disease. Inhibition of PAI-1 activity may have utility in the treatment of thromboembolic disease. We report here the evaluation of three diketopiperazine-based low molecular weight inhibitors of PAI-1 activity (XR334, XR1853 and XR5082). In vitro these compounds reversed the inhibitory effects of PAI-1 against both tPA and urokinase (UK) (IC50:5 to 80 μM). In contrast, other serpin-serine protease interactions, including α1-antitrypsin-trypsin, α2-antiplasmin-plasmin and antithrombin-thrombin, were not affected, neither did these inhibitors affect global tests of haemostasis. In the light of this promising in vitro profile these compounds were evaluated in a standard radioisotopic assay of clot lysis in whole rat blood following intravenous administration. In this assay these compounds dose-dependently enhanced fibrinolysis ex vivo. After intravenous bolus administration XR334, XR1853 and XR5082 at 5 mg/kg increased clot lysis by 32.0 ± 5.1% SEM (n = 25, p <0.01), 36.7 ± 3.5% SEM (n = 36, p <0.01) and 60.0 ± 2.8% SEM (n = 17, p <0.01) respectively compared to vehicle. Intravenous infusion of these compounds (1 mg/kg/min for 20 min) significantly prolonged (approximately twofold) the time to blood vessel occlusion in the rat electrically-stimulated carotid artery thrombosis model. Thus, these low molecular weight inhibitors of PAI-1 activity enhanced fibrinolysis ex vivo and protected against thrombus formation in the rat.
Development of Large Platelet Aggregates from Small Aggregates as Determined by Laser-light Scattering: Effects of Aggregant Concentration and Antiplatelet Medication
Authors: Tohgi Hideo , Takahashi Hiroaki , Watanabe Katsumi , Kuki Hiroyuki , Shirasawa Yoshiaki
Published: July 2018

Particle-counting methods that employ light scattering (LS) quantify changes in the number of platelet aggregates of different sizes after the application of an aggregating stimulus. Using the LS method, we studied the effects of aggregant concentration, aspirin administration, and ticlopidine administration on aggregate formation and compared the results with those obtained using the conventional optical density (OD) method. Subjects were 47 controls, 31 patients treated with aspirin (330 mg/day), and 37 patients treated with ticlopidine (200 mg/day). Platelet aggregation after stimulation by 0.5, 1.0 or 5.0 μM ADP, or 0.5, 1.0 or 2.0 μg/ml collagen was determined using both methods. Using the LS method, small (9-25 μm), medium (25-50 μm), and large (50-70 μm) aggregates were counted. In patients untreated with antiplatelet medication, greater concentrations of ADP or collagen generated larger aggregates. Generation of small and medium-sized aggregates showed a significant positive correlation with OD levels after stimulation with 0.5 or 1.0 μM ADP, or 0.5 or 1.0 μg/ml collagen. In patients treated with aspirin, the development of small aggregates into large aggregates was inhibited. Thus, the number of small aggregates increased. Inhibition induced by aspirin was more effective against aggregation after stimulation with collagen than with ADP. In patients treated with ticlopidine, small and medium-sized aggregate formation was inhibited after stimulation with low concentrations of ADP or collagen, but was promoted after stimulation with high aggregant concentrations. The capability of the LS method to quantify different sizes of aggregates after stimulation with low concentration agonists may facilitate investigation of the aggregation process, and of how this process is affected by antiplatelet agents.
Influence of Metabolic Control on Thromboxane Biosynthesis and Plasma Plasminogen Activator Inhibitor Type-1 in Non-insulin-dependent Diabetes mellitus
Authors: Davì Giovanni , Belvedere Mario , Vingneri Sergio , Catalano Isabella , Giammarresi Carlo , Roccaforte Salvatore , Consoil Agostino , Mezzetti Andrea
Published: July 2018

We have previously shown that tight metabolic control by insulin therapy reduced thromboxane-dependent platelet activation in noninsulin-dependent diabetes mellitus (NIDDM) patients. The present study was undertaken to determine whether a similar effect could be obtained without switching diabetics in secondary failure to insulin treatment. For this purpose, we gave strict diet and exercise advise program and adjusted on a weekly basis the oral antidiabetic therapy (glipizide) that 26 patients with NIDDM had been given over the previous months.

Basal measurements of urinary ll-dehydro-TXB2 and PAI-1 confirmed previous findings of enhanced levels of these parameters in NIDDM patients with macrovascular disease in comparison to age-and sex-matched controls. After 2-6 weeks, 16 patients achieved tight metabolic control associated with significant reduction of both thromboxane biosynthesis and PAI-1 levels; 10 patients remained in poor control and no significant decrease of both parameters was observed.

We conclude that reduction of in-vivo platelet activation and PAI-1 antigen levels after metabolic improvement obtained by frequent reassessment of sulphonylurea therapy together with strict diet and exercise programs may have beneficial effects on the progression of diabetic micro- and macrovascular disease.
Factor V Leiden, Antiphospholipid Antibodies and Thrombosis in Systemic Lupus Erythematosus
Authors: Fijnheer R , Horbach D A , Donders R C J M , Vilé H , Oort E v , Nieuwenhuis H K , Gmelig-Meijling F H J , de Groot Ph G , Derksen R H W M
Published: July 2018

Thromboembolic complications are frequently observed in patients with systemic lupus erythematosus (SLE). Significant associations have been reported between these complications and the presence of antiphospholipid antibodies, notably the lupus anticoagulant and anti-cardiolipin antibodies. Factor V Leiden is a genetic disorder associated with an increased risk of venous thrombosis. We studied these factors in 173 patients with SLE in relation to both arterial and venous thrombosis. The frequency of factor V Leiden in SLE patients is comparable to that in the Dutch population (5%) and a risk factor for venous thrombosis (odds ratio 4.9; Cl 1.2-19.6), but not for arterial thrombosis. The lupus anticoagulant is a risk factor for both arterial thrombosis (odds ratio 7.1; Cl 2.9-17.4) and venous thrombosis (odds ratio 6.4; Cl 2.7-15.4). From multivariate analysis, both the lupus anticoagulant and factor V Leiden appeared independent risk factors for venous thrombosis.
D-Dimer Test and Diagnosis of Deep Vein Thrombosis: A Comparative Study of 7 Assays
Authors: Elias A , Aptel I , Huc B , Chale J J , Nguyen F , Cambus J P , Boccalon H , Boneu B
Published: July 2018

The current D-Dimer ELISA methods provide high sensitivity and negative predictive value for the diagnosis of deep vein thrombosis but these methods are not suitable for emergency or for individual determination. We have evaluated the performance of 3 newly available fast D-Dimer assays (Vidas D-Di, BioMerieux; Instant IA D-Di, Stago; Nycocard D-Dimer, Nycomed) in comparison with 3 classic ELISA methods (Stago, Organon, Behring) and a Latex agglutination technique (Stago). One-hundred-and-seventy-one patients suspected of presenting a first episode of deep vein thrombosis were investigated. A deep vein thrombosis was detected in 75 patients (43.8%) by ultrasonic duplex scanning of the lower limbs; in 11 of them the thrombi were distal and very limited in size (<2 cm). We compared the performance of the tests by calculating their sensitivity, specificity, positive and negative predictive value for different cut-off levels and by calculating the area under ROC curves. The concordance of the different methods was evaluated by calculating the kappa coefficient. The performances of the 3 classic ELISA and of the Vidas D-Di were comparable and kappa coefficients indicated a good concordance between the results provided by these assays. Their sensitivity slightly declined for detection of the very small thrombi. Instant IA D-Di had a non-significantly lower sensitivity and negative predictive value than the 4 previous assays; however its performance was excellent for out-patients. As expected, the Latex assay had too low a sensitivity and negative predictive value to be recommended. In our hands, Nycocard D-Dimer also exhibited low sensitivity and negative predictive value, which were significantly improved when the plasma samples were tested by the manufacturer. Thus significant progress has been made, allowing clinical studies to be planned to compare the safety and cost-effectiveness of D-Dimer strategy to those of the conventional methods for the diagnosis of venous thrombosis.
Hemophilic Dog Model For Evaluating Hemostatic Efetcacy Of Plasma Protein Fractions
Authors: Kingdon H S , Hassell T M
Published: July 2018

About 15% of patients with hemophilia A develop inhibitors to Factor VIII. Because in many cases the inhibitor renders the patient refractory to treatment with Factor VIII, plasma protein fractions designed to bypass the inhibitor have been developed. In the USA these are referred to genetically as Anti Inhibitor Ccrplex Concentrates (AICCs). Development of AICCs has been hampered by lack of a suitable irodelin which to judge hemostatic efficacy. Similarly, lack pf a model has impeded research on the mechanism of action Of AICCs. Therefore, we chose to evaluate AICCs in dogs with hemophilia A, reasoning that a material capable of bypassing & Factor VIII inhibitor should be effective in Factor VIII deficient recipients with or without inhibitors. Under local anesthesia a standardized gingival biopsy was performed losing a flexible plastic template and a modified scalpel handle holding two #11 Bard-Parker blades. The parallel time is ions were 5 mm long, 2 nm apart, and 1.5 mm deep. The tissue block thus defined was removed by sharp dissection. In normal dogs, bleeding from this wound ceased in 5 ± 2min, the wound was filled with concave clot, and bleeding did not recur. In contrast, hemophilic dogs formed an abnormal (very large) clot, and rebled for several days if untreated. The hematocrit usually dropped by 2-10 percentage points in 24 hr of uncontrolled bleeding. An experimental AICC fraction under development by Cutter Laboratories was evaluated in 5 dogs, and shown to be hemostatically effective. The dose required to achieve hemostasis was 25-75 units/kg; in some dogs a second dose was required 6 hr after the first dose to maintain hemostasis. A single dog with a low titer inhibitor to Factor VIII was successfully treated with 39 u/kg, followed by a repeat dose of 39 u/kg 6 hr after the first dose. We conclude that this AICC preparation brings about nemostasis in Factor VIII deficient individuals with or without inhibitors to Factor VIII.
Cleavage Of Reduced Factor VIII/VON Willebrand Factor Subunits By Thrombin Or Factor Xa
Authors: Vehar G A
Published: July 2018

Purified Factor VIII/von Willebrand Factor (FVIII/vWF) was obtained from bovine plasma using ammonium sulfate and glycine precipitations, tricalcium citrate adsorption, and Sepharose CL-4B gel chromatography. The protein was concentrated with ammonium sulfate and dialyzed against 0.05 M Tris-HCl, pH 7.4, containing 0.15 M NaCl. Partial reduction of these preparations was obtained using a 2.5 hr incubation at 20°C with 5 mM dithioerythritol, followed by dialysis to remove the reductant. The addition of a 19 to 1 molar ratio (110 to 1 wt. ratio) of FVIII/vWF to thrombin resulted in a time dependent cleavage of the reduced preparation, as detected by SDS gel electrophoresis. A 50 min. digest resulted in the cleavage of the 220,000 dalton subunit to products of 145,000 and 140,000 daltons. Factor Xa, in the presence of calcium and phospholipid, also cleaved the reduced protein when present at a 14 to 1 molar ratio (75 to 1 wt. ratio) of FVIII/vWF to factor Xa . A 50 min. digest resulted in fragments of 200,000, 175,000, and 55,000 daltons. No cleavage occurred under these conditions if the native, unreduced FVIII/vWF was used. Factors XIIa, XIa, IXa and activated protein C did not cleave the reduced or unreduced preparations. The observed cleavages of FVIII/vWF by thrombin or factor Xa do not, however, correlate with the alteration of any known function of this protein complex.
GlycoSaminoglycan (GAG) Distribution In Catheter Induced Lesions In Rabbit Aortae In Relation To Lipid Accumulation
Authors: Moore S , Richardson M
Published: July 2018

Lipid-rich lesions develop in normolipemic rabbit aortae in response to continuing injury associated with an indwelling polyethylene catheter. Lipid disappears from these lesions when the catheter is removed. In contrast following removal of the endothelium by Fogarty balloon catheter, lipid accumulation, located mainly in areas of the neointima covered by regenerated endothelium, persists and increases over time. We have previously shown that this lipid accumulation, in areas of endothelial regeneration following balloon deendothelialization, is associated with an increase in GAG visualized by ruthenium red staining as large granules (RRG) seen on transmission electron microscopy in the interstitium of the neointima. Moreover there was a marked decrease in the concentration of large granules in the areas of neointima uncovered by endothelium where lipid accumulation was minimal or absent. In the present experiment, polyethylene catheters were placed in the aortae of rabbits and removed after 3 weeks. Early thrombi, raised lipid-containing lesions, flat lipid-free lesions and samples of normal aortae were examined at this time. Four weeks and 8 weeks following catheter removal, raised lesions, flat lesions and samples of normal aorta were examined. The GAG was quantitated by counting the RRG/square micron of intercellular space. Early, unorganized thrombi showed lipid- containing macrophages in the superficial part of the lesion and very few RRG (7±3/p2). Thrombi showing early organization without lipid vacuoles in the smooth muscle cells showed the content of RRG to be (52±19/μ2) half that of raised lesions containing lipid (115±13/μ2). Lipid-free lesions at 4 and 8 weeks following catheter removal showed less RRG granules (5±3/μ2) than control tissue (24±39/μ2) . Thus the accumulation and disappearance of lipid parallels the concentration of GAG in the interstitium of the neointima.
Glanzmann’S Thrombasthenia: Pregnancy And Parturition
Authors: Sundqvist S B , Nilsson I M , Svanberg L , Cronberg S
Published: July 2018

A 35-y-old patient with severe Glanzmann's thrombasthenia became pregnant. Only four such cases are so far known. Two of them were treated successfully with platelet concentrates. Our patient, who had received multiple transfusions, had antibodies against tissue antigens. These were of IgG type. PIPA-test (Kekomäki, R. Medical Biology 54:112, 1977) verified the presence of platelet antibodies. The second wave of aggregation induced by ADP in normal platelet-rich plasma was inhibited by her IgG fraction. Transfusion of HLA matched platelets did not result in any survival of the transfused platelets in her circulation, as revealed by the absolutely absence of aggregability even after the transfusion. Thus no specific treatment was available.

During the 24-32 weeks there were signs of activation of the coagulation and the fibrinolytic systems in the form of a decreasing number of platelets and the presence of FDP and fibrin monomers. VIII:C and VIIIR:Ag were remarkably low throughout the pregnancy and the fibrinogen content did not reach the upper limit of the reference area. The oestriol content in plasma and the biparietal diameter as measurements of the condition of the foetus, remained normal during this period, but there was an unexplainable decrease of the plasma oestriol content during the last month of pregnancy.

When signs of activation of the coagulation system had disappeared, tranexamic acid was given as prophylaxis (Astedt and Nilsson, Br Med J 1:756, 1978). Apart from transient haematuria in the 24th week of gestation, no complications were seen during the rest of the pregnancy.

In the 42nd gestational week the patient delivered a boy, spontaneously per vaginam. No abnormal bleeding occurred either during or after the delivery. Intense and prolonged drug induced contraction of the uterus might have helped to decrease the bleeding. No transfusion of platelets or blood was given.
Contact Activation By Ellagic Acid - The Concept Of Soluble Activator Disputed
Authors: Exner T , Rickard K A
Published: July 2018

Ellagic acid (EA) has often been described as a “soluble” activator of the contact mechanism in contrast to kaolin, silica or glass which are insoluble. We have found that EA is not effective as a contact activator when in true solution but is active only when present as a particulate solid or dispersion.

A pH titration of EA was carried out and it was found that EA dissolved readily in alkaline solutions. When adjusted to neutral pH these formed supersaturated solutions which slowly precipitated forming fine microcrystalline dispersions. At high EA concentrations there was concurrent development of turbidity and contact activity. At lower EA levels no turbidity was visible, yet contact activity increased on standing, presumably as EA particle size increased. Contact activity was reduced in all ellagic acid preparations by centrifugation after standing.

Substancesof low molecular weight in true solution do not sediment under these conditions and do not show turbidity. These are properties of particles or aggregates. These observations support the old theory that only particulate materials providing a liquid/negatively charged solid interface of substantial size function as true activators.

It is probably important to distinguish between activation of the contact mechanism by substances which may be in true solution such as various organic sulphates and those which are dispersions of solids, functioning possibly by a different mechanism.