bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Cloning, Characterization and Expression of cDNA for Rabbit Brain Tissue Factor
Authors: Pawashe Aruna , Ezekowitz Michael , Lin Tsung-Chung , Horton Renata , Bach Ronald , Konigsberg William
Published: July 2018

Tissue factor (TF) is a membrane anchored glycoprotein that initiates blood coagulation by forming a complex with circulating factor VII or VIIa. TF has been identified in atherosclerotic plaques and may possibly trigger thrombosis after spontaneous plaque rupture as seen in acute myocardial infarction or angioplasty. We have previously developed an atherosclerotic rabbit model for study of the acute and chronic outcomes following angioplasty. As a first step in developing inhibitors of TF, we have isolated and characterized a rabbit cDNA coding for the mature TF. The sequence comparison of rabbit TF cDNA with those of human and mouse TFs show considerable similarity at both the nucleotide and amino acid levels. The TF cDNA when expressed in E. coli demonstrates a procoagulant activity comparable to that of native rabbit brain TF. The TF activity can be blocked by a polyclonal antibody against rabbit TF.
125I-Fibrinogen Binding to Platelets in Myeloproliferative Disease
Authors: Mistry R , Cahil M , Chapman C , Wood J K , Barnett D B
Published: July 2018

Recent reports have suggested a variation in the density and affinity of fibrinogen binding sites in platelets from patients with myeloproliferative disorders (MPD) which may reflect platelet functional abnormalities in these subjects. We have investigated the binding of 125I-fibrinogen (125I-Fb) to gel-filtered platelets from a large relatively homogeneous group of patients with MPD compared to normal age matched controls. Twenty-two of the patients investigated had polycythaemia vera and four essential thrombocythaemia.

The maximal density and affinity (Kd) of 125I-Fb binding was assessed by saturation analysis in gel-filtered platelets (GFP) stimulated with either 10 εM ADP or 150 mU thrombin. In addition the functional significance of the binding sites was studied by evaluating the response of GFP from the two experimental groups by assessing the effects of increasing concentrations of added fibrinogen on the response to 10 εM ADP using standard light transmission aggregometry.

In both groups the density of fibrinogen binding sites expressed in response to thrombin stimulation was significantly higher (approximately 2-3 fold) than that found in response to ADP. However, fewer binding sites were detected in the MPD group as compared with the control group in response to both ADP and thrombin. The Kd for 125I-Fb was similar for both agonists in normal controls and was significantly lower than that found in the MPD subjects.

Although the 125I-Fb binding study results indicate a significant reduction in both the number and affinity of fibrinogen binding sites in patients with myeloproliferative disorders, the clinical and functional significance of these findings remain uncertain.
LY53857, a 5HT2 Receptor Antagonist, Delays Occlusion and Inhibits Platelet Aggregation in a Rabbit Model of Carotid Artery Occlusion
Authors: Wilson Harve C , Coffman William , Killam Anne L , Cohen Marlene L
Published: July 2018

The present study was designed to evaluate the effectiveness of the ergoline 5HT2 receptor antagonist, LY53857 in a rabbit model of vascular arterial occlusion. LY53857 (1 and 10 εM) inhibited serotonin amplified platelet aggregation responses to threshold concentrations of ADP in rabbit platelets in vitro. LY53857 (1 εM) not only inhibited the serotonin component of rabbit platelet aggregation, but also inhibited in vitro aggregation induced by ADP (48.7 ± 16.7% inhibition), collagen (76.1 ± 15.9% inhibition) and U46619 (65.2 ± 12.3% inhibition). The effectiveness of this ergoline 5HT2 receptor antagonist in blocking aggregation to ADP, collagen and U46619 may be related to its ability to inhibit a serotonin component of platelet aggregation since rabbit platelets possess high concentrations of serotonin that may be released during aggregation produced by other agents. Based on the effectiveness of LY53857 to inhibit rabbit platelet aggregation, we explored the ability of LY53857 to extend the time to carotid artery occlusion in rabbits following electrical stimulation of the artery. Reproducible carotid artery occlusion was induced in rabbits by moderate stenosis coupled to arterial cross clamping, followed by electrical stimulation. With this procedure, occlusion occurred at 47.0 ± 7 min (n = 30) after initiation of the electrical stimulation. Animals pretreated with LY53857 (50 to 500 εg/kg i.v.) showed a delay in the time to carotid artery occlusion (at 100 εg/kg i.v. occlusion time extended to 164 ± 16 min). Furthermore, ex vivo platelet aggregation from animals treated with LY53857 (300 εg/kg i.v.) resulted in 40.5% inhibition of platelet aggregation in response to the combination of ADP (1 εM) and serotonin (1 εM). These studies document the ability to obtain reproducible arterial occlusion in the rabbit and showed that intravenously administered LY53857 prolonged the time to carotid artery occlusion. Prolongation of carotid artery occlusion time was accompanied by inhibition of serotonin-amplified ADP-induced aggregation in rabbit platelets, an effect observed both in vitro and ex vivo. Thus, the rabbit is a useful model for studying the effectiveness of 5HT2 receptor antagonists in prolonging vascular occlusion induced by insult of the carotid artery.
The Role of GTP-Binding Proteins in Platelet Activation
Authors: Manning David R , Brass Lawrence F
Published: July 2018

Platelet activation begins with the binding of an agonist to the cell surface and culminates in the events of platelet aggregation, secretion and clot formation. Recent studies have identified two large families of GTP-binding proteins in platelets that are thought to participate in the events of platelet activation. The first of these are the G proteins, heterotrimeric proteins which are best known for their ability to mediate the interaction between agonist receptors and intracellular enzymes such as adenylyl cyclase, phospholipase C and phospholipase A2. To date, at least six G proteins have been identified in platelets: Gs, Gz, three variants of Gi and either Gq or G11 (or both). An additional, pertussis toxinresistant G protein, Gq, may also be present. The second group of GTP-binding proteins present in platelets is substantially smaller than the heterotrimeric G proteins, ranging in size from 21 to 28 kDa. At least 15 such low molecular weight GTP-binding proteins have been identified in platelets, many of which are homologous to the products of the ras proto-oncogenes. In cells other than platelets, low molecular weight GTP-binding proteins have been implicated in protein transport, cell activation events and malignant transformation. Their role in platelets is unknown.
Deep Vein Thrombosis and Fibrinolysis
Authors: Levi Marcel , Lensing Anthonie W A , Büller Harry R , Prandoni Paolo , Dooijewaard Gerard , Cuppini Stefano , ten Cate Jan Wouter
Published: July 2018

In the present study 57 consecutive patients with a first episode of venographically proven deep vein thrombosis were investigated to evaluate the release of tissue-type plasminogen activator (t-PA) and of urokinase-type plasminogen activator (u-PA) in response to DDAVP stimulation as well as the resting plasminogen activator inhibitor (PAI) concentration, comparing this to the results obtained in 66 similar patients with a clinical suspicion of thrombosis but with a normal venogram. All assays were performed without knowledge of the patient's status.

Four patients in the deep vein thrombosis-group (7%) had an absent u-PA antigen response upon DDAVP infusion, while a normal response was observed in all control subjects. Patients and controls showed similar increases in t-PA antigen level upon DDAVP. High resting PAI antigen levels were encountered in 5 patients in the deep vein thrombosis-group (9%) and in 6 subjects in the control group (9%).

The results from this controlled study indicate that a defective release of u-PA may occur in patients with deep vein thrombosis and may have pathogenetic significance. Furthermore it is concluded that elevation of PAI levels cannot be considered as a specific risk factor for venous thrombosis.
Low Molecular Weight Heparin-Catalyzed Inactivation of Factor Xa and Thrombin by Antithrombin III – Effect of Platelet Factor 4
Authors: Schoen Pieter , Lindhout Theo , Franssen Jo , Hemker H Coenraad
Published: July 2018

Low molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding material, so mean molecular weights as such might bear little information on their anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities by platelet factor 4 (PF4). These properties were investigated in pure systems with proteins of human origin. Pseudo-first order rate constants of inactivation of factor Xa and thrombin by antithrombin III were determined as function of heparin concentration, in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor Xa over the anti-thrombin activities were essentially the same for the 4th International Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32), CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed that the heparin molecules of the upper region of the molecular weight distribution are responsible for the anti-thrombin, but also to a large extent for the anti-factor Xa activities. The results indicate that depolymerization of unfractionated heparin does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the presence of Ca2+-ions the rate constants of inactivation of factor Xa are lowered as compared to those of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin activities of fixed concentrations of the LMW heparins was studied by measuring rate constants as function of PF4 concentration. All anti-thrombin and 50% of the anti-factor Xa activities were readily neutralized. Excess PF4 was required to neutralize another 35-50% of the anti-factor Xa activities. At PF4 levels obtained at maximal release of the content of platelet α-granules, all anti-thrombin and most (≥85%) of the anti-factor Xa activities can be neutralized.
Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity
Authors: Brandt John T
Published: July 2018

Lupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.
Detection of the Glanzmann's Thrombasthenia Mutations in Arab and Iraqi-Jewish Patients by Polymerase Chain Reaction and Restriction Analysis of Blood or Urine Samples
Authors: Peretz H , Seligsohn U , Zwang E , Coller B S , Newman P J
Published: July 2018

Severe Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews – an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs – a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.
Thrombin Generation and Fibrinolysis in the Thrombotic Thrombocytopenic Purpura and the Hemolytic-Uremic Syndrome
Authors: Monteagudo J , Pereira A , Reverter J C , Pijoan J , Tusell J , Puig L , Ordinas A , Castillo R
Published: July 2018

Prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complexes (TAT), as well as other coagulation and fibrinolysis parameters, were studied in a series of 13 patients affected by thrombotic thrombocytopenic purpura (TTP) or hemolytic-uremic syndrome (HUS). Fragment F1+2 was found to be increased in all patients at diagnosis (patients' range, 1.21-19.03 nmol/1; normal limits, 0.28-1.08 nmol/1), and remained also higher than normal after treatment with plasma exchange (patients' range, 1.5-4.01 nmol/1). Even though the analysis of fibrinolysis markers did not show a definite state of hypo or hyperfibrinolysis in the systemic circulation, enhanced circulating D-dimer levels (0.53-12.6 ug/ml, normal levels of 0.03-0.29 εg/ml) indicated that a certain grade of fibrin lysis was present at previously formed thrombi. Plasma PAI-1 activities either on admission (9.2-38.2 U/ml) and after plasma exchange therapy (2.6-38.6 U/ml) showed a behavior irrespective of t-PA: Ag changes, and post-plasmapheresis values remained high only in patients with fatal neurological outcome. Nevertheless, no correlations between clinical and laboratory data could be established useful for the TTP/HUS prognosis. We conclude that increased thrombin generation occurring in damaged areas is appropriately inhibited by antithrombin III in the systemic circulation, avoiding consumption coagulopathy to develop in uncomplicated patients. In addition, fibrinolysis data suggest that elevated PAI-1 may decisively favor the development of microvascular thrombi.
Lipoprotein Associated Coagulation Inhibitor, Factor VII, Antithrombin III, and Monocyte Tissue Factor Following Surgery
Authors: Carson Steven D , Haire William D , Broze George J , Novotny William F , Pirrucello Samuel J , Duggan Michael J
Published: July 2018

Fifteen patients undergoing major surgical procedures were evaluated for lipoprotein associated coagulation inhibitor (LACI) antigen, factor VII (F VII), antithrombin III (AT III), and peripheral blood monocyte tissue factor (TF) activity immediately before surgery and on following days. A peak in monocyte TF activity occurred between the first and fifth days after surgery in 10 of the patients, while LACI, F VII, and AT III levels dropped in a qualitatively parallel manner in 8 of these patients. LACI, F VII, and AT III levels decreased after surgery in two additional patients even though TF activity also decreased after surgery in these patients. In the remaining 3 patients who developed infections during the study, TF activity rose within 2 days of the diagnosis of infection in addition to the postoperative peak. In two of these patients, LACI levels increased dramatically near the end of the study period without concomitant changes in F VII and AT III. Overall, the presurgical TF levels in disrupted monocytes varied 52-fold and the maximal TF activity varied 24-fold among patients. The TF response following surgery is therefore heterogenous in both temporal occurrence and magnitude of the postsurgical peak. The patients also varied considerably in the presurgical levels of monocyte TF activity. A possible association between the level of presurgical TF activity and the magnitude of the postsurgical peak was noted. Thirteen of the patients had malignant disease, and, as a group, had presurgical levels of LACI (83 ± 26; mean ± SD), F VII (124 ± 22) and AT III (111 ± 14) within the normal ranges established with pooled reference plasma and similar values for three normal donors studied over the same period (AT III 121 ± 6, LACI 95 ± 4, and F VII 112 ± 29). No cause-effect relationship could be established between changes in TF activity and changes in the other factors measured. Patient- or therapy-specific variables may determine postoperative variations in LACI, F VII, AT III, and monocyte TF.
The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor
Authors: Teitel Jerome M
Published: July 2018

An experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.
Pharmacokinetic and Hemostatic Properties of the Recombinant Plasminogen Activator BM 06.022 in Healthy Volunteers
Authors: Martin U , von Möllendorff E , Akpan W , Kientsch-Engel R , Kaufmann B , Neugebauer G
Published: July 2018

In a randomized, single-blind, placebo-controlled, cross-over Phase-I study pharmacokinetic and hemostatic properties of BM 06.022 were investigated in seven healthy, male human volunteers. The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 domain and the protease domain of human t-PA and is unglycosylated due to its expression in Escherichia coli cells. Vehicle or 6 MU (= 10.4 mg) BM 06.022 was administered as a single i.v. bolus injection of 10 ml over 2 min. BM 06.022 was well tolerated. Fibrinogen levels and clotting times remained unchanged at baseline levels after 6 MU BM 06.022; plasminogen and α2-antiplasmin (collected on chloromethylketone) decreased maximally to 83 ± 1% and 64 ± 3%, respectively, of baseline. D-dimers and fibrinogen degradation products increased to 1,006 ± 234 ng/ml and 555 ± 155 ng/ml, respectively, after BM 06.022. Half-life of BM 06.022-activity was 11.2 ± 0.4 min and of antigen was 13.9 ± 0.7 min, followed by a terminal half-life only for antigen of 173 ± 33 min. Plasma clearance of BM 06.022 was 371 ± 13 ml/min for activity and 183 ± 15 ml/min for antigen. Thus, BM 06.022 is not fibrinogenolytic at 6 MU and is a fibrinolytic agent with a longer half-life than t-PA.
Abnormal Plasminogen Maywood I
Authors: Robbins K C , Boreisha I G , Godwin J E
Published: July 2018

Maywood I is a dysfunctional plasminogen. It is described in a patient (W. Y) with a reduced plasma functional activity and with a low normal antigen level. Plasminogen was isolated from the patients plasma by affinity chromatography with L-lysine-substituted Sepharose. The protein yield was 86 mg/1, which was 88% of the plasma Plg antigen level; the specific activity was 24.4 IU/mg protein compared to 28.5 IU/mg protein for the native molecule. The protein was the Glu-form determined by SDS-PAGE and by isoelectric focusing. Six major isoelectric forms were found with isoelectric points between pH's 6.40 and 5.45. Titration of the equimolar plasminogen.streptokinase complex with p-nitrophenyl-p-guanidinobenzoate gave 85% active-sites indicating a homogenous population of molecules; therefore, the propositus is a homozygote. Four different plasminogen activators: a) streptokinase, b) urokinase c) the plasmin-derived light (B) chain-streptokinase complex, and d) tissue plasminogen activator (with soluble fibrin/CNBr-fibrinogen fragments) generated little plasmin from the variant plasminogen (4.5 to 45 nM), 5% or less than that generated from normal plasminogen. At 45 nM plasminogen, the molar ratio of plasminogen : activator was 3.0 for streptokinase, 3.9 for urokinase, 7.1 for the light (B) chain-streptokinase complex, and 155 for tissue plasminogen activator. In the equimolar variant plasminogen.streptokinase complex, the active-site was slowly developed, to a maximum of 85% in 40 min; in the normal complex, 100% active-sites were developed in 15 min. The variant plasminogen forms two equimolar complexes with streptokinase (I and II), with different mobilities in PAGE, in about equal amounts. About 10% of complex I (abnormal), with a slower mobility than complex II (normal), converts to plasmin.streptokinase in 60 min, whereas about 40% of complex II converts to plasmin.streptokinase in 60 min. A kinetic defect was also found with this variant plasminogen. It showed substantially lowered kinetics of activation second-order rate constants with both streptokinase and urokinase, primarily due to lower catalytic rate constants, and with streptokinase, a higher Michaelis constant. Crossed-immunoelectrophoresis, and preparative isoelectric focusing isoelectric points did not show a charge mutation. Maywood I may be classified as a dysplasminogenemia with active center and kinetic defects, Type 1c.
Effects of Hirudin and Heparin on the Binding of New Fibrin to the Thrombus in t-PA Treated Rabbits
Authors: Agnelli Giancarlo , Pascucci Claudia , Cosmi Benilde , Nenci Giuseppe G
Published: July 2018

The aim of this study was to compare the ability of heparin and recombinant hirudin (r-hirudin) in preventing accretion of new fibrin on thrombi during and after treatment with tissue-type plasminogen activator (t-PA) and in enhancing t-PA induced fibrinolysis in a rabbit jugular vein thrombosis model. Heparin and r-hirudin were infused at doses capable of doubling aPTT. In the fibrin accretion inhibition experiments t-PA was infused over 3 h at a dose of 0.2 mg/kg along with saline or heparin, 0.75 mg/ kg or r-hirudin, 1.25 mg/kg. In rabbits treated with t-PA plus saline, heparin or r-hirudin, an accumulation of 125I-fibrinogen on the thrombi of 52.5 ±5.1 εg, 49.5 ± 5.6 εg and 23.5 ± 3.5 εg was observed, respectively, the difference between r-hirudin and both saline and heparin being statistically significant (p <0.01). The inhibition of fibrin accretion on the thrombi induced by r-hirudin persists for at least 9 h after the end of the infusion. By that time r-hirudin has been cleared from the circulation and aPTT has returned to the baseline level for at least 8 h. t-PA, 0.2, 0.4, and 1 mg/kg, infused with saline produced 34 ± 6%, 52 ± 5% and 79 ± 8% lysis of pre-formed thrombi, respectively. The same doses of t-PA infused with heparin, 0.75 mg/kg, produced 32 ± 3%, 54 ± 5% and 78 ± 6% fibrinolysis, respectively and infused with r-hirudin, 1.25 mg/kg, 38 ± 3%, 57 ± 5% and 82 ± 8%, respectively. Thus, no differences in fibrinolysis were observed among the groups of rabbits treated with heparin, r-hirudin and saline receiving the same dose of t-PA. When thrombolysis was assessed by thrombus weight treatment with t-PA and r-hirudin was more effective than treatment with t-PA and saline or heparin. The positive effects of r-hirudin on t-PA induced thrombolysis we observed deserve to be confirmed in clinical settings.
Hemostatic Responses to Mental Stress during the Menstrual Cycle
Authors: Jern Christina , Manhem Karin , Eriksson Elsa , Tengborn Lilian , Risberg Bo , Jern Sverker
Published: July 2018

To study the effect of sex hormones on the hemostatic responses to stress, blood samples were collected before, during, and after 20 min of mental stress from 9 healthy, non-smoking female volunteers, examined in the follicular and luteal phase. Mental stress caused significant increases in heart rate, blood pressure, and plasma catecholamines. In addition, analysis of variance indicated significant changes of leukocyte count, hematocrit, fibrinogen, von Willebrand factor antigen, t-PA activity and antigen in response to the stress test. However, in contrast to a male group previously investigated, there were no significant changes in factor VII coagulant activity in either menstrual phase. Overall the responses were more pronounced in the luteal as compared to the follicular phase. The findings support the concept that both gender and physiological variations in female sex hormones may modulate hemostatic responses to psychosocial stress.
Comparison of Real-Time B-Mode Ultrasonog raphy and Bilateral Ascending Phlebography for Detection of Postoperative Deep Vein Thrombosis Following Elective Hip Surgery
Authors: Borris Lars C , Christiansen Hanne M , Lassen Michael R , Olsen Agnete D , Schøtt Peder ,   (The Venous Thrombosis Group)
Published: July 2018

A prospective study compared real-time B-mode ultrasound examination with bilateral ascending phlebography in the diagnosis of postoperative deep vein thrombosis in 60 patients undergoing elective total hip replacement. The overall sensitivity and specificity of the ultrasound method were 54 and 91%, respectively, and the positive and negative predictive values were 83 and 69%, respectively. The rather low overall sensitivity of the ultrasound method in this study was due to difficulty in detecting thrombi smaller than 1 cm wherever they were located in the deep veins, and in diagnosing thrombi in the calf, regardless of their size. We conclude that real-time B-mode ultrasonography is a technique that can easily be used routinely for detection of postoperative DVT in hip surgery, but its sensitivity for proximal thrombosis (63%) is too low for it to be used alone.
Binding of Tissue Plasminogen Activator to Cultured Human Fibroblasts
Authors: Reilly Thomas M , Whitfield Mark D , Taylon David S , Timmermans Pieter B M W M
Published: July 2018

The binding of 125I-labeled, one-chain tissue plasminogen activator (t-PA) by WI-38 cultured human lung fibroblasts was investigated. Binding of t-PA to WI-38 monolayers was specific, saturable and temperature dependent. One and two-chain t-PAs were comparable in their ability to compete with 125I-labeled, one- chain t-PA for binding to fibroblasts, while no inhibition of binding was observed with a 500-fold molar excess of urokinase. Studies with various compounds suggest that neither the catalytic site, the fibrin binding site, nor the carbohydrate moieties on t-PA are involved in its binding to WI-38 cells. At higher temperatures, the amount of cell-bound 125I-t-PA that was removed by either incubation in binding buffer containing an excess of unlabeled t-PA, or by brief treatment with acidic buffer, was small (approximately 20%) suggesting that much of the t-PA is internalized. Electrophoretic analysis of extracts prepared from cells that had been incubated with 125I-t-PA revealed the presence of a major band of 70,000 Mr, which corresponds to intact t-PA. Our results suggest that WI-38 fibroblasts are capable of binding and internalizing t-PA, and that these processes involve a receptor site specific for t-PA.
Separation of Plasminogen Activators from Human Plasma and a Comparison with Activators from Human Uterine Tissue and Urine
Authors: Kok Preben
Published: July 2018

Normal human plasma contains acid-stable as well as labile plasminogen activators. The activity of activators in plasma euglobulins was inhibited by EACA in an uniform pattern, similar to that obtained with the major activators in human uterine tissue or with the purified porcine tissue activator, but different from the patterns obtained with plasmin or with urokinase.

Gel filtration at high ionic strength separated activators corresponding to particle sizes of 60,000 dalton and about 10,000 dalton, corresponding to two activators similarly obtained from human tissue. The 60,000 dalton activator was precipitated in the euglobulin fraction. Its concentration increased in plasma after exercise. The 10,000 dalton activator was found mainly in the supernatant. Gel filtration in 0.15 M solutions yielded activators in fractions of molecular sizes of 100-140,000 dalton and 200,000 dalton or larger.

The activity of normal and exercise euglobulins was inhibited by antiserum to a plasminogen activator prepared from porcine tissue, but it was not inhibited by antiserum to urokinase. Plasminogen activators in human plasma euglobulins resembled immunochemi- cally the activators in human uterine tissue.
Protein C Deficiency in Insulin-Dependent Diabetes: A Hyperglycemia-Related Phenomenon
Authors: Ceriello Antonio , Quatraro Antonio , Russo Patrizia Dello , Marchi Egidio , Barbanti Miriam , Milani Maria Rita , Giugliano Dario
Published: July 2018

In 30 insulin-dependent diabetic patients protein C (PC) antigen and PC activity were significantly lower than those of matched control healthy subjects. An inverse correlation between fasting plasma glucose and both PC concentration and activity was present in diabetics, while a direct correlation between PC concentration and PC activity was observed. Induced hyperglycemia in diabetic and normal subjects was able to decrease both PC antigen levels and PC activity, and heparin reversed in part this effect.

In diabetic patients euglycemia obtained by insulin infusion restored to normal the depressed PC levels. Heparin did not alter both the basal PC concentration and activity in healthy controls.

These data stress the major role of hyperglycemia in determining PC decrease in diabetics, and suggest that PC reduction is probably associated to hyperglycemia-enhanced thrombin formation.
Increased Monocyte Procoagulant Activity Independent of the Lupus Anticoagulant in Patients with Systemic Lupus Erythematosus
Authors: de Prost D , Ollivier V , Ternisien C , Chollet-Martin S
Published: July 2018

Monocytes can play a role in the activation of coagulation via increased procoagulant activity (PCA). We investigated the level of monocyte PCA in 19 patients with systemic lupus erythematosus (SLE), given the high rate of thrombotic events in this condition. Nine of these subjects also presented the lupus anticoagulant (LA). The PCA generated by patient monocytes was significantly higher than control values and was identified as tissue factor-like. Moreover, the number of monocytes with membrane-associated D dimer, a parameter whieh we have shown to be correlated with the PCA expressed in vitro by endotoxin-activated monocytes, was also significantly increased. Serum from both groups of patients (i.e. SLE and SLE + LA) stimulated the generation of PCA by control monocytes. By contrast, purified IgG from both patient groups had the same effect as control IgG on PCA generation by control monocytes. The nature of the stimulating agent in the serum was not identified. In conclusion, increased monocyte PCA may account for the increased incidence of thrombosis in SLE patients, although other, superimposed, factors would appear to exist in SLE + LA patients, given the higher incidence of thrombosis in this subgroup.
Subcutaneous vs Intravenous Heparin in the Treatment of Deep Venous Thrombosis – A Randomized Clinical Trial
Authors: Pini M , Pattacini C , Quintavalla R , Poli T , Megha A , Tagliaferri A , Manotti C , Dettori A G
Published: July 2018

271 patients with acute symptomatic deep venous thrombosis of lower limbs, confirmed by strain-gauge plethysmography and/ or venography, were randomly assigned to receive intermittent subcutaneous heparin calcium or heparin sodium by continuous intravenous infusion for 6–10 days. Heparin dosage was adjusted to maintain activated partial thromboplastin time values (Throm-bofax reagent) at 1.3–1.9 times the basal ones. Strain-gauge plethysmography was repeated at the end of heparin treatment, and evaluation of therapy was performed by comparing the indexes of venous hemodynamics and by assessing the incidence of pulmonary embolism and of bleeding complications.

In the intravenous group, Maximal Venous Outflow (MVO) increased from 20.8 ± 12.8 to 28.4 ± 17.5 ml/min per 100 ml of tissue and Venous Capacitance (VC) from 1.39 ± 0.92 to 1.94 ± 1.0 ml/100 ml of tissue (mean ± SD). In the subcutaneous group, MVO increased from 21.0 ± 12.7 to 27.5 ± 18.1 and VC from 1.60 ± 0.86 to 2.06 ± 1.0. The median improvement of MVO and VC were 22% and 36% respectively in the IV group and 20% and 24% in the SC group. Clinical pulmonary embolism occurred in 2 patients in the intravenous group (1 fatal) and in 4 in the subcutaneous group (1 fatal). 9 major bleeding complications occurred in the intravenous group (1 fatal) and 5 in the subcutaneous group (1 fatal). The differences were not significant at the statistical analysis.

The results suggest that subcutaneous intermittent heparin has a comparable efficacy to continuous intravenous heparin in the treatment of deep venous thrombosis.

To the same conclusion points an overview of the seven randomized trials which compared these treatment modalities.
Identification of Different Forms of Human C4b-Binding Protein Lacking β-Chain and Protein S Binding Ability
Authors: Hessing Martin , Kanters Deon , Hackeng Tilman M , Bouma Bonno N
Published: July 2018

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical α-chains (70 kDa) and one β-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the β-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silverstaining and with Western-blotting using monoclonal antibodies specific for the β-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 × 108 and 3.21 × 108 M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the β-chain and was capable of protein S binding (KA: 4.35 × 108 M-1). The two other forms of C4BP lacked the β-chain and were unable to bind protein S.
A Collaborative Study to Establish the Second International Standard for Streptokinase
Authors: Heath A B , Gaffney P J
Published: July 2018

An International Standard for Streptokinase - Streptodomase (62/7) has been used to calibrate high purity clinical batches of SK since 1965. An international collaborative study, involving six laboratories, was undertaken to replace this standard with a high purity standard for SK. Two candidate preparations (88/826 and 88/824) were compared by a clot lysis assay with the current standard (62/7). Potencies of 671 i.u. and 461 i.u. were established for preparations A (88/826) and B (88/824), respectively.

Either preparation appeared suitable to serve as a standard for SK. However, each ampoule of preparation A (88/826) contains a more appropriate amount of SK activity for potency testing, and is therefore preferred. Accelerated degradation tests indicate that preparation A (88/826) is very stable.

The high purity streptokinase preparation, coded 88/826, has been established by the World Health Organisation as the 2nd International Standard for Streptokinase, with an assigned potency of 700 i.u. per ampoule.
Heparin Effect on Bone Density
Authors: Ginsberg J S , Kowalchuk G , Hirsh J , Brill-Edwards P , Burrows R , Coates G , Webber C
Published: July 2018

In order to provide estimates of the risks of symptomatic osteoporosis and reduced bone density in premenopausal women treated with long-term (greater than 1 month) heparin therapy, we evaluated a cohort of 61 consecutive premenopausal women previously treated with long-term heparin (cases) and a group of controls matched for age, parity and duration between the last pregnancy and evaluation. All patients underwent dual photon absorptiometry of the lumbar spine and single photon absorptiometry of the wrist and most cases underwent plain lateral radiography of the thoracolumbar spine in order to exclude silent fractures. Although none of the cases suffered symptomatic fractures (0 of 61, 95% confidence intervals 0.0 to 5.9%), there was a significantly greater proportion of cases than controls with bone density below our pre-defined levels. The long-term implications of our findings are uncertain but because it is possible that the reduction in bone density predisposes women to fractures, this potential risk should be considered when treating women with long-term heparin.
Ristocetin and Botrocetin Involve Two Distinct Domains of von Willebrand Factor for Binding to Platelet Membrane Glycoprotein lb
Authors: Girma J P , Takahashi Y , Yoshioka A , Diaz J , Meyer D
Published: July 2018

We have evidence that ristocetin and botrocetin mediate binding of von Willebrand Factor (vWF) to platelet glycoprotein lb (GPIb) through two distinct domains on the vWF molecule. This was established by using monoclonal antibodies (MAbs) to vWF and synthetic peptides derived from the sequence of vWF. MAb 322 and MAb NMC/vW 4 both recognize native vWF as well as fragments containing the GPIb-binding domain of vWF, obtained with the following enzymes: trypsin (116 kDa), V-8 pro tease (Spill, 320 kDa) and V-8 protease plus subtilisin (33-28 kDa). Nevertheless, the lack of reciprocal displacement between the two MAbs in experiments of competitive inhibition for binding to vWF demonstrate that their respective epitopes are separate. Both MAbs inhibit 125I-vWF binding to platelet membrane GPIb and vWF-dependent platelet agglutination induced by ristocetin. However, only MAb NMC/vW4 inhibits these functions in the presence of botrocetin and when ristocetin-induced platelet agglutination is inhibited by MAb 322, botrocetin is still able to restore the agglutination. The involvement of two distinct domains of vWF for binding to GPIb in the presence of ristocetin or botrocetin was confirmed in experiments of binding of 125I-vWF to platelets using as competitor synthetic peptides corresponding to the GPIb binding domain of vWF (Cys 474 to Pro 488 and Ser 692 to Pro 708). At a final concentration of 2.5 mM both peptides inhibit more than 90% of the binding of vWF to ristocetin-treated platelets but are unable to modify this binding in the presence of botrocetin. In conclusion our data suggest that botrocetin and ristocetin involve distinct sites on vWF for binding to GPIb.
The Anticoagulant Mechanism of Action of Recombinant Hirudin (CGP 39393) in Plasma
Authors: Lindhout Theo , Blezer Ron , Coenraad Hemker H
Published: July 2018

We studied the inhibitory action of recombinant desulphatohirudin (CGP 39393) on thrombin generation in whole plasma. Human plasma was activated either with thromboplastin or factor IXa. Hirudin delayed thrombin generation, but it was unable to prevent the explosive appearance of thrombin. The
dose-dependent prolongation of the lag phase of the intrinsic and extrinsic thrombin generation curve was not the result of titration of thrombin activity by hirudin but the result of a delayed formation of the prothrombin converting complex (prothrombinase). In case of extrinsic activation, hirudin did not affect factor Xa generation, but prolonged the lag phase of the factor Va generation curve, causing its appearance when factor Xa generation was already in the decay phase. Because of its inhibitory action on the thrombin-mediated activation of factor VIII, hirudin prolonged the lag phase of the factor X converting complex that consists of factor IXa and factor VIIIa. Our observations with hirudin are in keeping with the notion that inhibition
of the thrombin-mediated amplification reactions in blood coagulation is a very efficient way to delay or inhibit completely thrombin generation. However, although hirudin neutralizes stoichiometric amounts of thrombin, the interaction between in situ generated thrombin and hirudin appears not to be fast enough to prevent trace amounts of thrombin to activate factors VIII and V. Consequently, an explosive thrombin generation is observed even when free hirudin is present.
Chlorobutanol, a Preservative of Desmopressin, Inhibits Human Platelet Aggregation and Release In Vitro
Authors: Chen Shih-Luen , Yang Wu-Chang , Huang Tung-Po , Wann Shiang , Teng Che-ming
Published: July 2018

Therapeutic preparations of desmopressin for parenteral use
contain the preservative chlorobutanol (5 mg/ml). We show here
that chlorobutanol is a potent inhibitor of platelet aggregation and release. It exhibited a significant inhibitory activity toward several aggregation inducers in a concentration- and time-dependent manner. Thromboxane B2 formation, ATP release, and elevation of cytosolic free calcium caused by collagen, ADP, epinephrine, arachidonic acid and thrombin respectively were markedly inhibited
by chlorobutanol. Chlorobutanol had no effect on elastase- treated platelets and its antiplatelet effect could be reversed. It is concluded that the antiplatelet effect of chlorobutanol is mainly due to its inhibition on the arachidonic acid pathway but it is unlikely to have a nonspecitic toxic effect. This antiplatelet effect of chlorobutanol suggests that desmopressin, when administered for improving hemostasis, should not contain chlorobutanol as a preservative.
Hypersensitivity to Thromboxane A2 in Cholesterol-Rich Human Platelets
Authors: Tomizuka Takuya , Yamamoto Kyohei , Hirai Aizan , Tamura Yasushi , Yoshida Sho
Published: July 2018

The effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,ll-epithio-ll,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.
Relationships of Plasma Viscosity, Coagulation and Fibrinolysis to Coronary Risk Factors and Angina
Authors: Lowe G D O , Wood D A , Douglas J T , Riemersma R A , Macintyre C C A , Takase T , Tuddenham E G D , Forbes C D , Elton R A , Oliver M F
Published: July 2018

Plasma viscosity, molecular markers of activated coagulation and fibrinolysis (fibrinopeptides A and Bβ15-42), coagulation factors (fibrinogen and factor VII) and antiplasmins were measured in 529 men aged 35-54 years and related to new angina pectoris (n = 117) and to coronary risk factors in controls without angina (n = 412). Five major risk factors (cigarette-smoking, blood pressure, cholesterol, triglyceride and body mass index) were each associated with increases in plasma viscosity, coagulation factors, and imbalance of coagulation over fibrinolysis (increased ratio of fibrinopeptide A/fibrinopeptide Bβ15-42). Increased viscosity and fibrinogen in smokers were partly reversed in exsmokers, but the imbalance of coagulation and fibrinolysis persisted. Cholesterol and triglyceride were also associated with increased antiplasmin activity. In men with angina, only fibrinogen was elevated compared to controls. We suggest that increased plasma viscosity and an imbalance of coagulation over fibrinolysis may be mechanisms by which known risk factors promote arterial thrombosis, but are not present in stable angina.
Assessment of Therapeutic Quality Control in a Long-Term Anticoagulant Trial in Post-Myocardial Infarction Patients
Authors: Azar Aida J , Deckers Jaap W , Rosendaal Frits R , van Bergen Paul F M M , van der Meer Felix J M , Jonker Jan J C , Briëf Ernest
Published: July 2018

Various methods have been described to evaluate efficacy of anticoagulant therapy using the international normalized ration (INR). We compared the following approaches: (1) total INR’s or the most recent measurement; (2) percent time within therapeutic range, with INR changing directly or halfway between visits; and (3) total observation time assuming INR changing linearly. The study population comprised 1700 post myocardial infarction patients. Treatment comprised 3725 patient-years. There were 61,471 INR assessments with target therapeutic level of 2.8–4.8. Acenocoumarol as well as phenprocoumon were employed. Therapeutic achievement in the first months of treatment was low: less than 60% of INR’s were in range. Treatment stabilized after 6 months. Patients on acenocoumarol were within range 70% of the time compared to 80% for phenprocoumon. Method 3 is preferred because it incorporates time and is capable of calculating incidence rates at different INR levels. Our findings call for an urgent improvement of standard of anticoagulant control in the first months following commencement of treatment.