bluuebunny/crossref_metadata_2025 huggingface dataset:
Tanakan Inhibits Platelet-Activating-Factor-Induced Platelet Aggregation in Healthy Male Volunteers
Authors: Guinot P. , Caffrey E. , Lambe R. , Darragh A.
Published: April 2009

An open study to investigate the PAF-acether antagonist activity of Tanakan in healthy male volunteers examined the effect of a single 15-ml oral dose on ex vivo platelet aggregation induced by adrenaline, adenosine diphosphate (ADP), collagen or PAF-acether. Aggregometry was performed on platelet-rich plasma samples from blood taken 1 h before dosing with Tanakan and 2, 4 and 8 h after intake of Tanakan. Following dosing with Tanakan there was a reduction in platelet aggregation at all doses of PAF, with 1 μM ADP and adrenaline, the most significant decreases occurred with 75 nM PAF-acether 4 h after intake (p < 0.05) and 300 nM PAF-acether 4 h (p < 0.01) and 8 after intake (p < 0.05). There were no concomitant changes in coagulation, skin bleeding time, haematological and biochemical laboratory tests, blood pressure or pulse. The results provide a possible explanation for the clinical efficacy of Tanakan in the treatment of peripheral vascular disease; they also confirm that a single oral dose of 15 ml Tanakan is well tolerated.
Catecholamines Do Not Induce Fibrinolytic Activity Increase in Cultured Bovine Endothelial Cells
Authors: Peracchia F. , Lipartiti M. , Fratelli M. , De Blasi A. , Donati M.B. , Mussoni L.
Published: April 2009

This study has investigated the catecholamine involvement in the fibrinolytic modulation of cultured bovine aortic endothelial cells (BAEC). Adrenaline and isoproterenol, at concentrations ranging from 10 to 100 μM, were unable to modulate the fibrinolytic response of these cells. β-Adrenergic binding studies using 3H-CGP 12177 as radioligand evidenced the presence of about 23,113 ± 2,065 sites/cell, with a KD of 1.23 ± 0.29 nM. Isoproterenol stimulated cAMP accumulation at concentrations ranging from 1 to 100 μM, with a maximal accumulation of 30 pmol/106 cell. Hence, in our experimental conditions BAEC, although possessing functional β-adrenergic receptors, were unable to increase any fibrinolytic activity in response to catecholamines.
Prophylaxis of Deep Venous Thrombosis with a Low-Molecular-Weight Heparin (Kabi 2165/Fragmin®) in Stroke Patients
Authors: Prins M.H. , Gelsema R.G. , Sing A.K. , van Heerde L.R. , den Ottolander G.J.H.
Published: April 2009

In a group of 60 patients in the acute phase of an ischemic stroke the assumption that low-molecular-weight heparin Kabi 2165 in the dose of 2 × 2,500 anti-Xa units s.c. could prevent thromboembolic complications was tested using a double-blind, placebo-controlled, randomized trial design. Thirty patients were allocated to each group. Both treatment groups were comparable with regard to neurological status and general condition. In the Kabi 2165 group there were 6 cases of deep venous thrombosis (DVT) compared to 15 in the placebo group (p = 0.05). In the placebo group there were 4 deaths during the trial versus 9 in the Kabi-2165-treated group (NS). Cerebral bleeding complicated 2 cases in the placebo group versus 4 in the Kabi 2165 group (NS). These results indicate that in ischemic stroke patients Kabi 2165 2 × 2,500 anti-Xa units s.c./24 h reduces the frequency of DVT. Because of the small number of patients it is impossible to evaluate the safety.
Bleeding and Thrombosis in Chronic Myeloproliferative Disorders: Relation of Platelet Disorders to Clinical Aspects of the Disease
Authors: Wehmeier A. , Scharf R.E. , Fricke S. , Schneider W.
Published: April 2009

Bleeding and thrombosis are a major cause of morbidity and mortality in myeloproliferative disorders (MPD). This study evaluates the relation between thrombohemorrhagic complications and platelet abnormalities in different subgroups of MPD. In 57 MPD patients thrombohemorrhagic complications occurred in 71 % of patients with polycythemia rubra vera and 50% of patients with osteomyelofibrosis and primary thrombocythemia but in only 29% of patients with chronic myelogenous leukemia. Increased β-thromboglobulin and platelet factor 4 plasma levels, platelet aggregation defects, and increased dispersion of the platelet volume distribution curve were most frequent in those subgroups where most serious thrombohemorrhagic complications were observed, and multiple platelet-related abnormalities were often found simultaneously. Fibrinopeptide A plasma levels were rarely elevated, however. Our results indicate that platelet abnormalities associated with bleeding and thrombosis are primarily determined by the clinical subgroup of myeloproliferative disease.
Effect of Physical Training on Thrombotic Tendency in Rats: Decrease in Thrombotic Tendency Measured by the He-Ne Laser-Induced Thrombus Formation Method
Authors: Yamamoto J. , Iizumi H. , Hirota R. , Shimonaka K. , Nagamatsu Y. , Horie N. , Morita S.
Published: April 2009

The effect of physical training on thrombotic tendency was assessed in rats. Exercise was done on a flat treadmill for 30 min at a rate of 1,400 m/h (submaximal speed), 5 times a week for either 1.5 or 3 months. The thrombotic tendency was measured by the He-Ne laser-induced thrombus formation method in microvessels of mesentery, i.e. measurement of the number of laser irradiations necessary to induce stasis of blood flow by occlusive thrombus formation. An increase in the number of irradiations necessary to induce occlusive thrombus formation was observed in arterioles, but not in venules after physical training for 1.5 and 3 months.
Comparison of Human Normal, Full-Term, Fetal and Adult Plasminogen by Physical and Chemical Analyses
Authors: Summaria L.
Published: April 2009

Human fetal plasminogen was isolated from fetal cord blood obtained from full-term normal newborns. Two fetal plasminogen preparations were characterized by physical analyses and compared to adult human Glu-plasminogen. The protein concentration of plasminogen in each full-term fetal plasma was approximately 50% of the concentration found in adult plasma. The specific activity of the isolated plasminogen from both full-term fetal plasmas was 28.8 ± 1.5 IU/mg protein, approximately the same as that of adult Glu-plasminogen. No significant difference was observed in the rate at which plasmin was generated from the normal fetal plasminogen and the adult Glu-plasminogen using streptokinase, urokinase, and tissue plasminogen activator. Electrophoretic analyses in an acrylamide gel/sodium dodecyl sulfate dissociating system showed that the fetal plasminogens and the adult Glu-plasminogen were the same molecular size. Analyses in an acrylamide gel isoelectric focusing system indicated that fetal and adult plasminogen both contained the same twelve isoelectric forms, however, there was a slight difference in the distribution of the isoelectric forms. The fetal and adult plasminogens both contained 792 ± 1 amino acid residues, and there were no significant differences in amino acid composition between the fetal and adult preparations. These comparisons indicate that normal, full-term, fetal and adult Glu-plasminogen are identical.
Effect of Steroids during Escherichia coli Endotoxinaemia in Rabbits
Authors: Østerud B. , Tindall A. , Olsen J.O.
Published: April 2009

A study was carried out on the effect of high doses of methylprednisolone to rabbits in combination with or after challenging them with Escherichia coli endotoxin. The animals given steroids simultaneously with the endotoxin or 2–6 h thereafter all had significantly higher white cell counts 24 h after endotoxin infusion. A second dose of endotoxin given 24 h after the first caused a rapid fall in white cell count, and no difference between the groups could then be observed 2 h later.The most striking effect was observed when methylprednisolone was administered simultaneously with the endotoxin. This caused a suppression of the clearance of endotoxin from the circulation, and this in turn could probably account for some newly synthesized thromboplastin expressed by the circulating monocytes. Furthermore, monocyte activation appears to be significantly higher in the animals receiving steroids than in the controls when the animals are subjected to two doses of endotoxin injections 24 h apart. Thus, high doses of methylprednisolone cannot be seen to have any beneficial effect as judged from the parameters tested in the present study.
Importance of Factor-IX-Dependent Prothrombinase Formation – The Josso Pathway – in Clotting Plasma
Authors: Xi Ma , Béguin S. , Hemker H.C.
Published: April 2009

We report a study on the importance of factor IX activation in thromboplastin-dependent coagulation in plasma. Diluted, CaCl2-containing thromboplastin solutions at constant phospholipid concentration were used to trigger the coagulation in plasma from patients with congenital factor IX and factor VIII deñciency in the presence and absence of added factors IX and VIII, and the generation of thrombin activity was monitored. When coagulation is triggered with the high thromboplastin concentrations normally used in clinical routine tests, the generation of thrombin activity in plasma of patients with congenital factor IX deficiency before and after reconstitution with purified factor IX appears identical. When, however, coagulation is triggered with low thromboplastin concentrations, a clear dependency of the generation of thrombin activity on the concentration of factor IX becomes evident at factor IX concentrations lower than 30 nM (about 40% clotting factor activity). Factor VIII is a compulsory cofactor for this factor IX activity because the prothrombinase activity at optimal factor IX concentration is still critically dependent upon the amount of factor VIII present. The lower the amount of thromboplastin, the higher the importance of factor IX and factor VIII activation in thromboplastin-dependent coagulation. This suggests a role of this pathway in pathophysiological thrombin formation.
Thromboembolic Manifestations and Congenital Factor V Deficiency: A Family Study
Authors: Manotti C. , Quintavalla R. , Pini M. , Jeran M. , Paolicelli M. , Dettori A.G.
Published: April 2009

A case of congenital factor V deficiency is reported. Despite this defect in blood coagulation, the patient had experienced recurrent thrombophlebitis and was referred to us because of deep venous thrombosis of the lower limbs associated with pulmonary embolism. Both functional and immunological assays documented a deficiency of factor V (12 and < 10%, respectively). The available family members were investigated and the same defect was found in 2 brothers of the propositus, who also suffered from thrombotic diseases (recurrent thrombophlebitis and myocardial infarction). The propositus has been treated with long-term oral anticoagulant therapy, no hemorrhagic complications or thrombotic recurrences being recorded in 2 years’ time.
Congenital Factor XIII Deficiency with Multiple Benign Breast Tumours and Successful Pregnancy with Substitutive Therapy
Authors: Boda Z. , Pfliegler G. , Muszbek L. , Tóth A. , Ádány R. , Hársfalvi J.H. , Papp Z. , Tornai I. , Rak K.
Published: April 2009

A 34-year-old woman with congenital factor XIII (FXIII) deficiency and multiple connective tissue tumours is reported. The subunit a of FXIII was totally absent in her plasma, platelets and histiocytes of breast fibroadenomas and considerably reduced in the monocytes (below 5%). The plasmatic level of subunit b was also reduced (25%). She had a bleeding tendency and habitual abortions. Fresh frozen plasma therapy permitted a successful pregnancy.
Inborn Errors of Metabolism Causing Homocysteinemia and Related Vascular Involvement
Authors: Skovby Flemming
Published: April 2009

Sulfur amino acids have been implicated in the pathogenesis of thromboembolic vascular disease, and observations of patients with several inborn errors of metabolism have led to the ‘homocysteine theory of arteriosclerosis’. Homocysteine is an intermediate in the transsulfuration pathway and it enters into several other reactions, some of which involve transfer of methyl groups. An abnormally high concentration of homocysteine in the blood causes homocystinuria. Deficiency of cystathionine β-synthase is the most frequent cause of homocystinuria. Patients with this disorder are at risk for early vascular occlusions. Treatment with vitamin B6 of patients who are biochemically responsive to this vitamin reduces the risk of thromboembolism. Clinical or pathologic evidence of early vascular disease has also been provided in patients with homocysteinemia due to deficient (re)methylation of homocysteine to methionine. This may be caused by a deficiency of 5,10-methylenetetrahydrofolate reductase or by a deficient synthesis of cobalamins.
Clinical and Laboratory Features of Homocystinuria
Authors: Cacciari E. , Salardi S.
Published: April 2009

Classic homocystinuria is an autosomal recessive metabolic disease due to a cystathionine-β-synthase deficiency with consequent blocking of homocysteine and serine condensation for producing cystathionine. The characteristic biochemical abnormalities in the blood and urine are: abnormal accumulation of methionine, abnormal presence of homocystine and low values of cystathionine, cysteine or cystine (disulfide of the cysteine). The most frequent clinical signs are: subluxation of the lenses, mental retardation of different degrees, long bones excessively lengthened, scoliosis, susceptibility to arterial and venous thromboembolism. The latter is frequent after surgery, and may be life-threatening. This disease must be differentiated from Marfan’s syndrome via laboratory tests.
Mechanisms of Thrombogenesis and Accelerated Atherogenesis in Homocysteinaemia
Authors: Wilcken David E.L. , Dudman Nicholas P.B.
Published: April 2009

Thrombogenesis and accelerated atherogenesis occur in the homocystinurias, both those due to recessively inherited cystathionine β-synthase deficiency and to disorders of remethylation of homocysteine to methionine. The evidence strongly implicates high levels of plasma homocysteine as the mediator. Homocysteine damages cultured human venous and arterial endothelial cells and enhances detachment from their substrate, changes not found with comparable concentrations of other amino acids tested. Homocysteine is oxidized in vitro to homocystine in an oxygen-dependent reaction producing hydrogen peroxide. Since the effects of homocysteine in cell cultures can be prevented by catalase, hydrogen-peroxide-induced injury may be the mechanism responsible. Five different laboratories have documented an association between mild homocysteinaemia and premature vascular disease. The majority of affected patients are heterozygotes for cystathionine β-synthase deficiency whose endothelial cells may have an enhanced susceptibility to injury by homocysteine. Mild homocysteinaemia also occurs in chronic renal failure in which vascular disease is prominent. Mechanisms linking mild homocysteinaemia and possible vascular effects are not yet understood, but could involve prostaglandins and oxidized low-density lipoprotein, and possibly also free radicals.
Lowered Antithrombin III Activity and Other Clotting Changes in Homocystinuria: Effects of a Pyridoxine-Folate Regimen
Authors: Palareti Gualtiero , Coccheri Sergio
Published: April 2009

Few studies have dealt with blood-clotting changes in patients affected by homocystinuria. The aim of this contribution is to briefly review studies published so far on the topic and report the results of our investigation performed in 3 patients. At baseline we found reduced antithrombin III and factor VII levels in all the patients, in line with the results of other authors, and other slight and less constant changes such as lowered factor X activity and protein C antigen, and increased β-thromboglobulin levels. During pyridoxine and folate treatment, antithrombin III activity rapidly returned to normal; factor VII increased and β-thromboglobulin decreased. These blood-clotting abnormalities may play a role in the thrombotic tendency associated with homocystinuria. Their nature is still uncertain, but the improvement observed during active metabolic treatment suggests that the defect in amino acid transsulfuration of homocystinuria may directly affect synthesis or activity of some clotting factors.
The Italian National Research Council Project on Homocystinuria
Authors: Totani Licia , Donati Maria Benedetta
Published: April 2009

The Italian National Research Council (CNR) has launched a special project aiming at gathering competences on rare diseases, their pathogenesis and their possible pharmacological management. The subproject on homocystinuria has three main objectives: to establish the prevalence and incidence of the disease in the Italian population, to develop sensitive assays of sulfurated amino acids which allow discrimination of heterozygotes from normal subjects and to better define the complex interactions between homocystinuria and vascular complications.
Fibrinogen Barcelona II: A New Case of Aα 16 Arg → His Substitution
Authors: Borrell M. , Vila Ll. , Solá J. , Coll I. , Fontcuberta J.
Published: April 2009

We describe a new congenital dysfibrinogenemia: fibrinogen Barcelona II in 8 members of a family with no major bleeding or thrombotic tendency. Incubation of this fibrinogen with thrombin at low concentration releases half of the expected normal fibrinopeptide A (FPA) and with some delay it releases an abnormal FPA. Abnormal FPA was purified and sequenced and showed a change in the normal amino acid sequence: arginine in position 16 has been substituted by a histidine. This is another case of dysfibrinogenemia in which Aα 16 Arg → His has been identified as the cause of abnormal behavior of the fibrinogen molecule.
Reduced Levels of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor in Plasma of Patients with Familial Amyloidotic Polyneuropathy
Authors: Olofsson Bert-Ove , Nilsson Torbjörn K.
Published: April 2009

Tissue plasminogen activator (tPA) antigen levels and plasminogen activator inhibitor (PAI) activity were measured in the plasma from 23 patients with familial amyloidotic polyneuropathy. Both tPA levels and PAI activity were reduced to about half normal, even when the effects of patient age and body mass index were taken into account. There were higher mean tPA levels and PAI activity in patients with mild disability than in those with moderate or severe disability, but these differences were not statistically significant. There were normal tPA levels after venous occlusion of the upper arm, indicating a normal capacity of vascular endothelium to release tPA. This is the first reported disorder with reduced rather than increased basal tPA and PAI levels. The pathogenetic and clinical significances of these disturbances so far remain obscure.
New Possibilities for Platelet Shape Change Evaluation Using the Laser Rheoaggregometer
Authors: Jung Georges , Schultz Guy
Published: April 2009

The study of aggregation functions in blood platelets can at present be undertaken in vitro by measuring variations in intensity of transmitted light or electrical impedance change. These two methods allow only a general approach to the aggregation process. The laser rheoaggregometer was constructed to study early morphological modifications induced when the platelets are activated before aggregation. The results obtained are in keeping with the laws of light scattering and allow the measurement of platelet shape change.
Increased Macrophage Procoagulant Activity but Normal Endothelial Thrombomodulin in Rabbits Fed an Atherogenic Diet
Authors: Semeraro N. , Montemurro P. , Giordano D. , Pasquetto N. , Curci E. , Triggiani R. , Colucci M.
Published: April 2009

We have studied the procoagulant activity (PCA) of blood and spleen mononuclear phagocytes and the thrombomodulin activity of aortic segments in rabbits fed an atherogenic diet for 6 weeks as compared to rabbits fed a standard diet. Blood monocytes expressed negligible basal PCA (i.e., PCA measured immediately after cell isolation) both in treated and control rabbits. PCA induced by endotoxin in vitro was not different in the two groups. In contrast, dietary treatment resulted in a significant increase in the basal PCA of spleen cells (p < 0.01). Moreover, the latter produced significantly more PCA than control cells (p < 0.002) in response to endotoxin in vitro. The thrombomodulin activity associated with aortic endothelium was not different in the two groups of animals despite the presence of visible fatty streaks on the aortic endothelium of treated rabbits. When rabbits were given a single injection of endotoxin, spleen mononuclear phagocytes harvested 60 min after the injection from treated animals expressed three times more PCA (p < 0.01) than did cells from controls. In all instances PCA was identified as tissue factor. Endotoxin injection did not affect the thrombomodulin activity of thoracic aorta from both control and diet groups. It is suggested that dietary fats may cause early functional changes in mononuclear phagocytes that lead to an increased PCA production both in vivo and in vitro. These data may be relevant to an understanding of the role of monocytes-macrophages in the pathogenesis of atherosclerosis.
Platelet Functions in Recombinant Hirudin-Anticoagulated Blood
Authors: Glusa Erika , Markwardt Fritz
Published: April 2009

The influence of genetically engineered recombinant hirudin (r-hirudin) on platelet functions was studied. Depending on the concentration, r-hirudin inhibits the thrombin-induced aggregation and 14C-serotonin secretion to the same extent as native hirudin. Comparative studies in blood anticoagulated by r-hirudin, heparin or citrate show a significantly lower spontaneous platelet aggregation in r-hirudinized blood. The extent of the ADP-induced aggregation is nearly the same in r-hirudinized, heparinized or citrated plasma. In r-hirudinized plasma, however, aggregation is reversible. In contrast to heparinized or citrated plasma, adrenaline causes only a very slight aggregation in r-hirudinized plasma. ADP- or adrenaline-induced secretion of 14C-serotonin do not occur in r-hirudinized plasma. The collagen- as well as the PAF-induced aggregation and 14C-serotonin release in hirudinized plasma do not differ significantly from those in heparinized or citrated plasma. r-Hirudin is a suitable anticoagulant for studying platelet functions because it does not produce any alterations in platelet reactions and does not provoke any changes in the ionized calcium concentration in blood.
Tumor Cells Augment the Factor Xa-Catalyzed Conversion of Prothrombin to Thrombin
Authors: Sakai Toshiyuki , Noguchi Masashi , Kisiel Walter
Published: April 2009

Activation of blood coagulation and local fibrin deposition may contribute to tumor metastasis. We have examined the ability of four human tumor cell lines (COLO 205, HepG2, J82 and CAPAN-2) to augment the conversion of prothrombin to thrombin by factor Xa and calcium in the presence and absence of exogenous factor Va. Using a chromogenic substrate assay to assess thrombin formation, we observed that all the above cell lines accelerated prothrombin activation in the absence of exogenous factor Va. The order of effectiveness was COLO 205 > HepG2 > J82 > CAPAN-2. In the absence of cells, no detectable thrombin formation occurred. Pretreatment of COLO 205 and HepG2 cells with anti-human factor V IgG inhibited prothrombin activation in a dose-dependent manner, but was without effect in J82 and CAPAN-2 incubation mixtures. Factor V coagulant activity was observed in COLO 205 and HepG2 cells as well as their culture media, but was not detected in J82 and CAPAN-2 cells or their culture media. Biosynthetic labeling and immunoprecipitation studies revealed that COLO 205 and HepG2 cells constitutively synthesized factor V or a factor-V-like molecule that comigrated with human factor V/Va on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All four tumor cell lines exhibited saturation binding of exogenous human factor Va resulting in a dose-dependent enhancement of their ability to augment prothrombin activation. Our results indicate that these tumor cells can readily assemble a functional cell surface prothrombinase complex that may be important in fibrin deposition associated with the growth and metastatic progression of these, and perhaps, other tumors.
Interaction of the Combined Medication with the New Low-Molecular-Weight Heparinoid Lomoparan® (Org 10172) and Acenocoumarol
Authors: Stiekema J.C.J. , de Boer A. , Danhof M. , Kroon C. , Broekmans A.W. , van Dinther Th.G. , Voerman J. , Breimer D.D.
Published: April 2009

A high intravenous dose of the low-molecular-weight heparinoid Lomoparan® (Org 10172) was administered to 6 healthy males in a steady state of anticoagulation (Thrombotest) by acenocoumarol. Prothrombin time, activated partial thromboplastin time and Stypven time were prolonged to a degree which was greater than that expected on the base of the summation of the effects by each drug alone. This effect was observed for a period of up to 1 h. The Thrombotest was affected for up to 5 h after the intravenous administration of Org 10172, therefore it is deemed unsuitable for monitoring the combined effects of these two anticoagulants during this period. Acenocoumarol did not affect the pharmacokinetic parameters of Org 10172 with the exception of a slight reduction of the clearance of plasma anti-Xa activity.
Biochemical Characteristics of Fibrolase, a Fibrinolytic Protease from Snake Venom
Authors: Ahmed N.K. , Tennant K.D. , Markland F.S. , Lacz J.P.
Published: April 2009

Fibrolase, a fibrinolytic enzyme isolated from Agkistrodon c. contortrix (southern copperhead) venom, solubilizes fibrin primarily by rapid hydrolysis of the alpha and beta chains. Fibrolase is also an Aα, Bβ fibrinogenase. The breakdown products of fibrin and fibrinogen following incubation with fibrolase were different from those observed with plasmin. This enzyme is a metalloprotease that was inhibited by ethylenediaminetetraacetic acid. Fibrolase was inhibited by dithiothreitol, suggesting that disulfide bonds are important for catalytic activity. It was also inhibited by α2-macroglobulin, but not by the soybean or lima bean trypsin inhibitors, diisopropylfluorophosphate, or p-hydroxymercuribenzoate. Unlike thrombolytic agents such as streptokinase, fibrolase does not activate plasminogen as evidenced by the use of plasmin-specific chromogenic substrate S-2251 and by sodium do-decyl sulfate-polyacrylamide gel electrophoresis.
Variation in Surface Platelet Glycoprotein lb Expression in Bernard-Soulier Syndrome
Authors: Poulsen Lene O. , Taaning Ellen
Published: April 2009

Seven patients with Bernard-Soulier syndrome (BSS) and 15 presumed heterozygotes of BSS from four families are presented. Evaluation of their platelet membranes was performed by an enzyme-linked radioimmunosorbent assay (ELISA) technique including monoclonal antibodies specific for glycoprotein (GP) lb and GPIIb/IIIa. Analyses of platelets from the patients revealed 6–33% of the normal GPIb concentration; siblings showed nearly equal amounts of this component. One of the relatives had 44% of the normal level, while 85–98% (mean 92%) of the normal GPIb content was observed in the remaining relatives. Normal or slightly elevated levels of GPIIb/IIIa were detected in the patients as well as in the relatives. All relatives had normal platelet count, size distribution, bleeding time, and ristocetin-induced platelet aggregation. The patients and their relatives may account for a biological variation in GPIb expression or may represent a variant type of BSS.
Platelet-Derived Mitogenic Activity and Bone Marrow Fibrosis in Myeloproliferative Disorders
Authors: Romano Mario , Viero Piera , Cortellazzo Sergio , Barbui Tiziano , Donati Maria Benedetta , Poggi Andreina
Published: April 2009

Mitogenic activity, measured as 3H-thymidine incorporation by NIH 3T3 cells, following stimulation with platelet-rich-plasma-derived serum (PRS), platelet-poor-plasma-derived serum and platelet extract was studied in 14 patients with myeloproliferative disorders (MPD) and 7 normal subjects. Reduced mitogenic activity was found in PRS and platelet extract of patients with MPD, as compared to controls. The average levels of platelet-derived growth factor (PDGF) equivalents were as follows: 16.3 ± 7.2 pg/l06 platelets in controls, 6.2 ± 2.2 pg/l06 (p < 0.05) platelets in patients with polycythaemia vera, 1.8 ± 0.4 pg/106 (p < 0.01) platelets in patients with idiopathic myelofibrosis and 4.0 ± 0.8 pg/l06 (p < 0.05) platelets in patients with essential thrombocythaemia (Dunnett test). A reduction of intraplatelet levels of β-thromboglobulin, although not statistically significant, was found in the same patients. No apparent relation was found between the amount of PDGF equivalents and the degree of bone marrow fibrosis.
Attempts to Standardize Platelet Aggregation Measurements: Simplified Screening Tests
Authors: Kirchhof B. , Kirchhof U. , Etscheid G.
Published: April 2009

We present a standardized measurement of collagen and ADP-induced platelet aggregation with the help of a personal computer. The calculator improves the recording of measurements and the automatic calculation of parameters to define the strength of aggregation in various aggregation phases, as well as aggregation and disaggregation velocities. The dose-effect relationships between aggregants and five calculated parameters after addition of ADP and four after addition of collagen are demonstrated in the light of measurements taken in 40 healthy persons. Comparison of these measurements with those of 8 volunteers 24 h after they had taken 250 mg of acetylsalicylic acid and those of 5 patients with typical illnesses associated with hypo- and hyperaggregability shows that analysis of the aggregation with two reagent concentrations (1,2 and 4 μg/ml collagen, 1, 2 and 4 μmol/ml ADP) and the resulting values allow one to distinguish between normal, hypo- and hyperaggregable platelets.
Monitoring Therapy with LMW Heparin: A Comparison of Three Chromogenic Substrate Assays and the Heptest Clotting Assay
Authors: Abildgaard Ulrich , Norrheim Laila , Larsen Anne E. , Nesvold Anders , Sandset Per Morten , Ødegaard Ole Rasmus
Published: April 2009

Three LMW heparins (LMWH), one unfractionated heparin (UH), and international standards of LMWH and UH were compared in three chromogenic substrate (CS) assays and the ‘Heptest’ clotting assay. With a two-stage CS assay, linear standard curves were obtained in the 0.1–1.0 U/ml range, nearly coinciding for all preparations. With the one-stage CS assays, standard curves were curvilinear and similar for UH and the LMWH groups. In the Heptest assay, standard curves were linear for UH but not for LMWH. Mean recovery of LMWH, added to patients’ plasma samples was 70–98% for the four assays. Variation between individual recoveries was much greater with Heptest (coefficient of variation (CV) 35–44%) than with one-stage CS assays (CV 14–21 %) or two-stage CS assays (CV 7–8 %). For monitoring LMW heparin therapy, CS assays seem preferable to Heptest. The two-stage CS assay had superior accuracy, but the one-stage CS assays were easier to perform.
Comparison of Various Screening and Confirmatory Tests for the Detection of the Lupus Anticoagulant
Authors: Forastiero R.R. , Falcon C.R. , Carreras L.O.
Published: April 2009

Several assay systems have been proposed for detection of the lupus anticoagulant (LA). We compared several screening and confirmatory tests used for the detection of LA in 108 patients. LA was detected in 52 plasmas. The activated partial thromboplastin time (APTT) and the dilute Russell viper venom time (DRVVT) were the most sensitive screening tests as compared to the kaolin clotting time (p < 0.001). The platelet neutralization procedure in both the APTT and DRVVT systems was superior to APTT performed with high phospholipid concentration (p < 0.01) and tissue thromboplastin inhibition (p < 0.001) as confirmatory tests. There was an association between the presence of LA and antiphospholipid antibodies detected by the enzyme-linked immunosorbent assay (p < 0.001). In summary, our results show that APTT may be a sensitive test for the detection of LA when an appropriate reagent is employed, and that freeze-thawed platelets are more effective than phospholipids to neutralize LA activity.
Effect of Disulfiram on the Platelet Function and Fibrinolysis in Healthy Volunteers
Authors: Gleerup G. , Boström° S. , Hansson G. , Teger-Nilsson A.-C. , Sjöquist P.O. , Winther K.
Published: April 2009

Disulfiram was studied for platelet and fibrinolytic activity in 12 healthy volunteers of both sexes (age 23–75 years). Placebo was given for 7 days, followed by disulfiram, 800 mg for 2 days and 400 mg for an additional 12 days. Finally, there was another placebo period of 14 days. With the exception of an initial platelet activation on day 2, no significant effects were found on the platelet variables studied: platelet aggregation with collagen, ADP and adrenaline, β-thromboglobulin and platelet factor 4. Treatment for 14 days with disulfiram resulted in a decreased euglobulin clot lysis time: from 421 ± 82 to 246 ± 41 min (p < 0.01). After an initial increase, plasminogen activator inhibitor activity was slightly decreased on disulfiram, from 8.4 ± 1.6 on placebo to 6.0 ± 1.2 U/ml (p < 0.05) after 14 days of treatment. Plasminogen, fibrinogen and α2-antiplasmin were unchanged. It is concluded that disulfiram can increase fibrinolytic activity in healthy subjects.
Coagulation Activation in Diabetes mellitus
Authors: Van Wersch J.W.J. , Westerhuis L.W.J.J.M. , Venekamp W.J.R.R.
Published: April 2009

One hundred and fourty-eight insulin-dependent diabetic patients were available for this study; 56 males and 92 females. For the investigation of coagulation activation we determined activated partial thromboplastin time, thrombin time, and fibrinogen besides fibrin monomers and thrombin-antithrombin III complexes (TAT-III). We assessed large percentages of increased fibrinogen levels but non-significant increases of the mean values in comparison with the reference group. The values for thrombin time were significantly prolonged, although relatively small percentages were exceeding the reference range. For the activated partial thromboplastin time, the values exceeded the upper reference limit, and the mean values were significantly higher than those of the reference group. Also for the fibrin monomers we obtained often enhanced values, and, moreover, the values were significantly higher as compared with the reference subjects. The amount of TAT-III concentrations above the reference range was much smaller than for the fibrin monomers and the TAT-III levels were not significantly enhanced. The results presented here are indicative of coagulation activation in diabetics, as indicated by the fibrin monomers and more or less by the TAT-III levels. Moreover, there could be demonstrated a positive correlation between fibrin monomer levels and HbA1 concentrations.
Development of a Sensitive and Rapid Chromogenic Factor IX Assay for Clinical Use
Authors: Wagenvoord R. , Hendrix H. , Tran T. , Hemker H.C.
Published: April 2009

A chromogenic factor IX assay is developed which requires only two time-dependent steps. Diluted plasma is mixed with a reagent containing factors VIII and X. The reaction is started by addition of a reagent containing factor XIa, thrombin, CaCl2, and phospholipids. Then factor XIa activates factor IX if present, thrombin activates factor VIII, and subsequently the complete factor X activating complex (factor IXa, factor VIIIa, Ca ions, and phospholipids) rapidly activates factor X. Finally, ethylenediaminetetraacetic acid plus a chromogenic substrate are added to stop the reaction and to measure formed factor Xa. Factor Xa formation is proportional to the plasma factor IX concentration (from 0 to 140%). The two reagents needed for the assay are stable at room temperature during a whole working day and for 3 h at 37 °C. A new isolation procedure for factor VIII is described. Factor VIII is purified from bovine plasma in a few steps with a yield of 20% and a 8,000-fold purification.
Further Evidence that the Residual vWf:Ag in Porcine FVIIL·C Induces Human Platelet Aggregation
Authors: Saniabadi A.R. , Marney Y. , Belch J.J.F. , Lowe G.D.O. , Barbenel J.C. , Madhok R. , Forbes C.D.
Published: April 2009

Porcine or bovine factor VIII concentrates (FVIIl·C) have been used during the past 3 decades to control bleeding in patients who have developed antibodies to human factor VIII. Since current preparations of animal FVIIl·C are not known to transmit infectious agents such as hepatitis or human immunodeficiency virus, they are of potential therapeutic interest. A purified porcine FVIIl·C (Hyate:C) is now widely used as an alternative to human FVIIl·C in patients with inhibitor. Unlike earlier preparations of porcine FVIIl·C, thrombocytopaenia is rare with the current preparation. Nonetheless, it causes the aggregation of human platelets in vitro. Our aim was to identify precisely the plasma factor which induces platelet aggregation. The effects of commercial porcine FVIIl·C, porcine fibrinogen, porcine fibronectin and the corresponding preparations from human origin on platelet aggregation were studied. Platelet aggregation was quantified by measuring the fall in single platelet count in human whole blood. Of these preparations, only porcine FVIIl·C (0.1–1 U/ml) and porcine fibrinogen (80–600 μg/ml) induced a fall in single platelet count of up to 85% due to aggregation. The extent of aggregation was directly proportional to the amount (0.007–0.1 U/ml test aliquot) of residual von Willebrand factor antigen (vWl·Ag) in the preparations. A monoclonal antibody to vWl·Ag inhibited the aggregation. We believe that the aggregation of human platelets induced in vitro by porcine FVIIl·C is mediated by vWl·Ag which also may be responsible for thrombocytopaenia reported following administration of porcine FVIIl·C in vivo.
Preparation of Factor-VIII-Depleted Plasma with Antibodies and Its Use for the Assay of Factor VIII
Authors: Rothschild C. , Amiral J. , Adam M. , Meyer D.
Published: April 2009

Standardized factor VIII (FVIII)-defιcient plasma is necessary for the clotting assay of this protein. Ethical considerations and prevalence of HIV seropositivity in hemophilia A patients have required the replacement of severe hemophilia A plasma by another specific substrate for FVIII clotting assays. FVIII-deficient plasma was prepared by immunodepletion using polyclonal anti-von-Willebrand factor (vWF) and monoclonal anti-FVIII antibodies coated to agarose beads. This plasma, deficient in both FVIII and vWF, allows the evaluation of FVIII activity with a detection limit of 1 % as compared to normal plasma. This immunodepleted plasma was compared to plasma from severe hemophilia A for the FVIII determination in plasma from normal subjects, severe and mild hemophiliacs, patients with von Willebrand disease and dicoumarol-treated patients. In all cases, similar values were obtained with both reagents at various levels of FVIII, with a correlation coefficient of 0.994. Such a good correlation was also obtained for the assay of FVIII in concentrates and in plasma from hemophiliacs following infusion. This immunodeficient plasma thus represents a suitable reagent for the FVIII clotting assay and provides an accurate alternative to the use of hemophilic plasma.
Biological and Thrombolytic Properties of Fibrolase -A New Fibrinolytic Protease from Snake Venom
Authors: Ahmed N.K. , Gaddis R.R. , Tennant K.D. , Lacz J.P.
Published: April 2009

Fibrolase, a direct-acting fibrinolytic enzyme has been shown to cleave primarily the Aα and Bβ chains of human fibrin. We have previously reported that fibrolase also exhibits fibrinogenolytic activity and acts mainly as an α-chain fibrinogenase. In contrast to the action of streptokinase (plasminogen activator), fibrolase does not activate plasminogen. In vitro thrombolytic efficacy of fibrolase was determined by monitoring the release of radiolabel from iodinated fibrin and human blood clots. Fibrolase effectively digested the clots in a dose-dependent manner. The in vivo efficacy of fibrolase was evaluated in an animal model of arterial thrombosis. Fibrolase was found to be efficacious at dissolving femoral arterial clots following a single intravenous bolus administration. Time to reperfusion was dose dependent and similar to that observed with streptokinase. No adverse effects on blood pressure and heart rate were observed.
Evidence for a Reduced Heparin Cofactor II Biological Activity in Diabetes
Authors: Ceriello Antonio , Quatraro Antonio , Dello Russo Patrizia , Marchi Egidio , Milani Maria Rita , Giugliano Dario
Published: April 2009

A reduction of heparin cofactor II (HCII) biological activity, despite its normal plasma concentration, is reported in insulin-dependent diabetic patients. A good linear correlation between HCII activity and concentration is present in normal controls but not in diabetics. In these subjects HCII activity correlates inversely with fasting blood glucose and glycated proteins but not with Hb A1. These data demonstrate the presence of a depressed HCII activity in the presence of its normal plasma concentration in insulin-dependent diabetics and suggest a role for short-term metabolic control in conditioning this phenomenon.
Reliability of Phlebography in the Assessment of Venous Thrombosis in a Clinical Trial
Authors: Picolet H. , Leizorovicz A. , Revel D. , Chirossel P. , Amiel M. , Boissel J.P.
Published: April 2009

In the frame of a multicenter controlled study comparing the efficacy of low-molecular-weight heparin to standard heparin in the prevention of postsurgical thrombosis, 94 phelbograms were centrally evaluated by two independent radiologists. Three months after the first central evaluation, a new reading was performed with the same radiologists, and discrepancies were adjudicated by a senior radiologist. The number of discrepancies between the first and the second evaluation was high: 33 interpretations (35%) had a least one difference, including 14 (14.9%) discrpeancies regarding the main issue, i.e. the presence of venous thrombosis. After the adjudication by the senior radiologist, this number decreased but was still large: 22 films (23.4%) with at least one discrepant item in all and 11 related to the presence of venous thrombosis. This report shows that venous thrombosis assessed by phlebography, which is usually considered as a golden standard in clinical trials, deserves a thorough quality control procedure.
Physiology of Blood Coagulation
Authors: Bloom A.L.
Published: April 2009

Blood coagulation forms part of an integrated series of haemostatic reactions, involving plasma, platelet, and vascular components. Platelets adhere to damaged endothelium or to subendothelium under the influence of adhesive proteins, and when activated they aggregate and expose binding sites for coagulation factors. Platelets, therefore, act as vehicles to concentrate and potentiate coagulation reactions on the damaged vessels. Following interaction of the ‘contact’ factors XII and XI, the coagulation protease zymogens undergo sequential activation, resulting in the generation of thrombin, the conversion of fibrinogen to fibrin, and the formation of a platelet-fibrin haemostatic plug. The fibrinolytic system interacts to regulate fibrin deposition and removal during healing. Central to coagulation is the generation of thrombin. It is involved in promoting haemostatic reactions as well as a number of protective functions. The activities of thrombin and other serine proteases are modulated by the serine potease inhibitors (serpins), including antithrombin III and heparin cofactor II which are important in regulating the physiological anticoagulant action of glycosaminoglycans at the endothelium and the pharmacological action of heparin. Reduction of the formation or function of thrombin and other serine proteases is one of the primary aims of anticoagulant therapy.
Physiological Role of Vessel Wall Related Antithrombotic Mechanisms: Contribution of Endogenous and Exogenous Heparin-Like Components to the Anticoagulant Potential of the Endothelium
Authors: Preissner K.T.
Published: April 2009

The intact vessel wall provides diverse anticoagulant mechanisms which, together with circulating components, are actively and dynamically involved in the regulation of the haemostatic system. These mechanisms are able to specifically and co-operatively counteract various procoagulant inducers at localized sites, such that under physiological conditions the endothelium provides a non-thrombogenic surface. Upon activation of the haemostatic system, these different components help to limit the extent of the prothrombotic response of blood coagulation at the site of injury and thereby contribute on different molecular levels to the overall anticoagulant potential of the endothelium. Heparin and possibly vessel wall related glycosaminoglycans may modulate or even enhance some of these anticoagulant mechanisms. A detailed understanding of the molecular architecture of these different processes in vitro may thereby lead to possible new therapeutic regimens.
Pathogenesis of Thrombosis
Authors: Prentice Colin R.M.
Published: April 2009

The pathogenesis of thrombosis involves consideration of two processes: atherosclerosis and thrombosis. Atherosclerosis is a prolonged process in which interaction of lipids and the haemostatic system produces focal lesions at sites of turbulence in arteries. These lesions may rupture, precipitating the complication of thrombosis and leading to complete occlusion of the vessel, causing myocardial infarction, stroke, or acute ischaemia. In veins the atherosclerotic process does not occur, and thrombosis is precipitated by immobility often associated with accidental or surgical trauma. The changes in platelets, coagulation, fibrinolytic systems leading to atherosclerosis and thrombi are reviewed. The role of platelets and coagulation changes in these processes are emphasized by the beneficial effects of therapy, both antiplatelet and anticoagulant therapy. Last, the concept of hypercoagulability leading to accelerated arterial disease and the clinical value of laboratory tests used in its detection are considered.
Heparin Structure
Authors: Casu B.
Published: April 2009

Heparins used in therapy are largely constituted by sequences of the trisulfated disaccharide L-iduronic acid-2-sulfate→D-glucosamine-N,6-disulfate. These regular sequences are interrupted by undersulfated (occasionally, oversulfated) sequences containing D-glucuronic acid and N-acetylated D-glucosamine. Different heparin sequences are binding domains for heparin cofactors and plasma proteins. The active site for antithrombin is a specific pentasaccharide sequence containing 3-O-sulfated D-glucosamine. Heparin cofactor-II binds, less specifically, mostly to the regular sequences. The conformational flexibility of iduronic acid residues contributes to the binding versatility and to the ‘biological reactivity’ of heparin.
Mode of Action of Unfractionated and Low Molecular Weight Heparins on the Generation of Thrombin in Plasma
Authors: Hemker H.C. , Béguin S.
Published: April 2009

Heparins, unfractionated and low molecular weight, act primarily by their scavenging of thrombin (S-type heparins). Via the feedback effect on factor VIII this has a secondary effect on prothrombin conversion in the intrinsic pathway (activated partial thromboplastin time). The anti-Xa action of a heparin will not significantly inhibit prothrombin conversion, except in the case of ultra low molecular weight heparins (P-type heparins) that have no significant antithrombin activity. These P-type heparins need, therefore, be given at high doses to have an antithrombotic effect. In platelet-rich plasma heparins retard platelet activation by lowering thrombin levels. Activated platelets neutralize up to 0.5 U/ml of unfractionated heparin, but low molecular weight heparin is much less affected.