bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Cloning and Expression of Rabbit Heparin Cofactor II: A Plasma Thrombin Inhibitor Highly Conserved between Species
Authors: Sheffield William P , Schuyler Philip D , Blajchman Morris A
Published: July 2018

Heparin cofactor II (HCII), a circulating plasma protein that inhibits thrombin, is a member of the serine proteinase (serpin) family of proteins. The extent to which HCII structure is conserved actross species lines was investigated, by obtaining cDNA clones encoding rabbit HCII. Overlapping clones corresponding to rabbit HCII were obtained by the combined use of hybridization screening of a rabbit liver cDNA library, and by rapid amplification of cDNA ends (RACE). The consensus sequence obtained spans 2178 nucleotides, and is comprised of a 5' untranslated region of 77 nucleotides, an open reading frame of 1440 nucleotides, and 3' untranslated region of 661 nucleotides that concludes with a poly A tract. The open reading frame is subdivided into a secretory signal sequence of 19 amino acids, and a mature protein of 461 amino acids. Within the region comprising the mature protein, 87% of the amino acid residues are identical to those seen in human HCII. Expression of an appropriately modified form of the rabbit HCII clone in an in vitro reticulocyte expression system yielded two major polypeptides, of 60 and 56 kD respectively, both of which were able to form SDS-stable complexes with human α-thrombin, in a reaction accelerated by dermatan sulphate. The remarkable degree of homology observed between rabbit HCII and its human conterpart, indicating a high degree of conservation of structure through evolution, suggests an important function of HCII in hemostatis.
In Vivo Effect of Suloctidil as an Antiplatelet Agent
Authors: Stelzer Marcia R , Burns Thomas S , Saunders Robert N
Published: July 2018

The relationship between the effects of suloctidil in vivo as an antiplatelet agent and in vitro as a modifier of platelet serotonin (5-HT) parameters was investigated. Suloctidil was found to be effective in reducing platelet aggregates formation in the retired breeder rat as determined using the platelet aggregate ratio method (PAR) with an ED50 of 16.1 mg/kg 24 hours post administration. In contrast to the hypothesis that 5-HT depletion is involved in the anti-aggregatory mechanism of suloctidil, no correlation was found between platelet 5- HT content and this antiplatelet activity. Reduction of platelet 5-HT content required multiple injections of high doses (100 mg/kg/day) of suloctidil. Suloctidil administration for 8 days at 100 mg/kg/day, which lowered platelet 5-HT content by 50%, resulted in no permanent effect on ex vivo platelet 5-HT uptake or thrombin-induced release, nor alteration in the plasma 5-HT level. However, these platelets exhibited a short-lived, significant increase in percent leakage of 5-HT after 30 minutes of incubation. Therefore, suloctidil treatment at high doses may with time result in platelet 5-HT depletion, however this effect is probably not related to the primary anti-aggregatory activity of the drug.
Effect of Factor XII Deficiency on Pregnancy and Parturition
Authors: Saidi Parvin , Siegelman Martin , Mitchel Valerie B
Published: July 2018

The clotting parameter of a primigravida with factor XII deficiency was studied during her third trimester of pregnancy, labor and post-partum; and compared with those of her newborn male infant. Sharp increases in factors VII, VIII, IX, X and moderate increases in factors II and XI were documented during pregnancy and at labor. All factors had returned to normal or near normal levels 24 hours after delivery. Factor XII remained at 0.0 level throughout. In the infant the clotting factor levels reflected depression of vitamins Independent factors II, IX and X and a factor XII level of 40.0%. No undue bleeding was noted in the mother at delivery or placental separation, and no bleeding manifestation was apparent in the infant. These findings suggest that factor XII does not play a major role in triggering or modulating the course of normal labor, nor is its absence necessarily associated with bleeding complications during parturition or placental separation.
Fibrinogen: Agreement of Experimental and Calculated Hydrodynamic Data with Electron-Microscopic Models
Authors: Serrallach Eugene N , Hofmann Victor E , Zulauf Martin , Binkert Thomas , Hofmann Robert , Känzig Werner , Straub P Werner , Schwyzer Robert
Published: July 2018

There is no general agreement on the size and shape of the fibrinogen molecule. We have studied the diffusion of the fibrinogen in solution by means of dynamic light scattering, nanosecond fluorescence depolarization and analytical ultracentrifugation. The results obtained under physiological concentration, pH an ionic strength are DT = 2.0 × 10-7 cm2sec-1, DR⊥ = 40’000 sec-1. Nanosecond fluorescence depolarization yielded DR// = 1.6 × 106 sec-1. Tentatively this value is interpreted as DR//, namely rotational diffusion about the major axis of the molecule. The sedimentation coefficient is 8.1 S. The hydrodynamic parameters derived from our measurements were compared with those calculated on the basis of the models proposed by Hall and Slayter, Hudry-Clergeon and Marguerie et al., Bachmann and Lederer, and Köppel.

The agreement is poor even if the degree of hydration is varied within wide limits. However, satisfactory agreement can be achieved by assuming a flexible molecule of about 900 Å length corresponding to two end-to-end bound trinodular structures of the Hall and Slayter type, with a nodule diameter of about 45 Å. Experimental evidence indicates that the discrepancies between the different models might be due to different techniques of sample preparation leading to different conformations of the molecule.
Is the Antithrombotic Activity of “Antiplatelet” Drugs Based on Protection of Endothelium?
Authors: Hladovec Josef
Published: July 2018

In only five “antiplatelet” agents has the antithrombotic activity been confirmed both clinically and experimentally. These include acetylsalicylic acid, dipyridamole, clofibrate, hydroxychloroquine and sulfinpyrazone. Whereas only acetylsalicylic acid has a demonstrable effect on platelet aggregation in doses usually applied, the effects of all remaining drugs are more easily explained by their influence on the vessel wall. This assumption is supported by the finding that all five drugs possess a stabilizing effect on endothelium in doses corresponding closely to the clinical dose range. The method based on counting detached endothelial cells in blood after a standard stimulus was used to demonstrate this effect.
Ionophoretic Activities of Phospholipids on Human Platelets
Authors: Ikeda Yasuo , Kikuchi Masao , Toyama Keisuke , Watanabe Kiyoaki , Ando Yasuhiko
Published: July 2018

The effects of phospholipids on calcium uptake by human platelets were investigated utilizing 45CaCl2. Among the phospholipids tested, cardiolipin and phosphatidic acid exerted a significant increase of calcium uptake by platelets. Calcium uptake was dependent upon cardiolipin concentration and incubation time. These phospholipids enhanced platelet aggregation induced by ADP (2 μM) or epinephrine (0.5 μg/ml). Cardiolipin caused the significant release of serotonin from platelets in the absence of externally added calcium in the medium.

These results may suggest that phospholipids activate platelets through their ionophoretic activities.
The Polymerase Chain Reaction with Sequence Specific Primers for the Detection of the Factor V Mutation Associated with Activated Protein C Resistance
Authors: Kirschbaum Nancy E , Foster Paul A
Published: July 2018

The prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by MnII digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.
Antithrombotic Action of Endogenous Porcine Protein C Activated with a Latent Porcine Thrombin Preparation
Authors: McBane Robert D , Wysokinski Waldemar E , Chesebro James H , Owen Whyte G
Published: July 2018

Endogenously activated protein C is evaluated for antithrombotic activity in porcine carotid arteries subjected to mechanical trauma. Protein C is activated by intravenous administration of guanidinobenzoyl- thrombin, which binds to thrombomodulin and there deacylates to yield thrombin. The bound, transiently active thrombin yields a peak of anticoagulant activity between 5 and 10 min after infusion of the latent thrombin. Inhibition of thrombin binding in vivo by co-infusing an active-site-blocked thrombin preparation elicits acute and lethal systemic thrombosis. Nearly occlusive platelet thrombosis, which occurs within 30 min of crushing 1 cm segments of carotid arteries with a standard hemostat, is blocked by endogenous protein C activation initiated 2 min before the crush injury. It is concluded that activated protein C blocks thrombosis in deeply injured musculo-elastic arteries, and that activation of latent thrombin bound to thrombomodulin in vivo is a practical means for delivery of pharmacologically effective concentrations of activated protein C.
Kinetics of the Inhibition of Tissue Factor-Factor Vila by Tissue Factor Pathway Inhibitor
Authors: Lindhout Theo , Franssen Jo , Willems George
Published: July 2018

Tissue factor-factor VIIa catalysed activation of factor X and factor IX is inhibited by the complex of tissue factor pathway inhibitor (TFPI) and factor Xa. At present, no information is available as to what extent the kinetics of complex formation between TFPI and factor Xa during factor X activation contribute to the overall rate of inactivation of the factor X converting complex. We have determined the kinetic parameters of the individual reactions, i. e. factor X activation, formation of the TFPI-factor Xa complex, and inactivation of tissue factor-factor VIIa by the TFPI-factor Xa complex. We modelled the overall reaction by assuming a two-step reaction: factor Xa generated by tissue factor-factor VIIa forms a reversible complex with TFPI and in the second step this complex forms a reversible quaternary complex with tissue factor- factor VIIa. The validity of the model was demonstrated by analysis of factor Xa generation curves in the presence of TFPI. Independently determined constants for factor X activation (kcat= 12 s-1, Km = 70 nM) and inhibition of tissue factor-factor VIIa by TFPI-factor Xa complex (rate constant of inhibition of 1.1 × 108 M-1s-1) were used. The association rate constant of the formation of the TFPI-factor Xa complex was estimated by fitting the model to the data. The rate constants of association of the complex between factor Xa and the variants full length TFPI, TFPI 1-247 and TFPI1-61 were very close to the values determined independently in a kinetic study on the inhibition of factor Xa in the presence of phospholipids, namely 3.4 × 106 M-1s-1, 0.4 × 106 M-1s-1 and 0.3 × 106 M-1s-1, respectively. These results indicate that the factor Xa-dependent inhibition of tissue factor-factor VIIa-catalysed factor X activation by TFPI can be adequately described by the two-step reaction sequence. We found that phospholipids (25 mol % phosphat-idylserine/75 mol % phospatidylcholine) increased the rate constant of association with factor Xa for full length TFPI, but not for the C-ter- minus truncated TFPI. Our results further indicate that optimal inhibition of tissue factor-factor VIIa activity is obtained with full length TFPI because of the higher rate of TFPI-factor Xa complex formation.
Inherited Platelet δ-Storage Pool Disease in Dogs Causing Severe Bleeding: An Animal Model for a Specific ADP Deficiency
Authors: Callan Mary Beth , Bennett Joel S , Phillips Deborah K , Haskins Mark E , Hayden James E , Anderson Jamie G , Giger Urs
Published: July 2018

The nature of a disorder producing moderate to severe bleeding after minor trauma, venipuncture, and surgery was studied in 3 families of American cocker spaniel dogs. In the 5 affected dogs tested, platelet counts and measurements of plasma coagulant function and von Willcbrand factor were normal. However, bleeding times were prolonged in 4 of the 5 affected dogs tested, and platelet aggregation in response to ADP and collagen was consistently abnormal in 3, suggesting that the bleeding disorder was due to abnormal platelet function. Measurements of 14C-serotonin uptake and retention by the affected platelets were normal. However, their ADP content was decreased, while their ATP content was normal, resulting in a mean ATP/ADP ratio of 8.32, compared to a mean ratio of 1.9 in normal canine platelets. Electron microscopy revealed that the number and appearance of the dense granules in the affected platelets were indistinguishable from those of normal controls. These studies suggest that this bleeding disorder results from a deficient δ-granule storage pool of ADP; given the normal serotonin uptake and retention by affected platelets and the apparently normal number of dense granules, the ADP deficiency may be the consequence of a selective defect in δ-granule ADP transport. Additional studies of this unique platelet disorder will provide an opportunity to understand the mechanism of adenine nucleotide storage in platelet δ granules.
Interaction of Echicetin with a High Affinity Thrombin Binding Site on Platelet Glycoprotein GPIb
Authors: Peng Manling , Emig Frances A , Mao Ahua , Lu Weiqi , Kirby Edward P , Niewiarowski Stefan , Kowalska M Anna
Published: July 2018

Echicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin (<0.2 U/ml), by binding to platelet glycoprotein lb (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin (>0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.
The Effect of Fibrin Clots and Clot-Bound Thrombin on the Development of Platelet Procoagulant Activity
Authors: Kumar Rachana , Béguin Suzette , Hemker H Coenraad
Published: July 2018

We tested different types of clot for their ability to provoke procoagulant activity in platelets: normal clots from platelet poor plasma (des AABB- or fibrin II clots), similar clots in which the adsorbed thrombin has been inhibited by hirudin, and clots obtained by the action of two snake venom enzymes that release only fibrinopeptide A (des AA- or fibrin I clots). Analogous clots from fibrinogen solutions were also tested.

In platelet rich plasma (PRP), where platelet coagulant phospholipids (PCP) are rate limiting for thrombin generation, the addition of any type of clot enhances the generation of thrombin thus it induces the appearance of PCP. Clots containing active adsorbed thrombin are the most potent ones in this respect. Lactate dehydrogenase (LDH) levels do not increase in the course of the thrombin generation so the platelets are not damaged in the process. Non-centrifugable PCP could be demonstrated to appear during the process, so the production of procoagulant phospholipid microparticles must be part of the mechanism. Membrane transbilayer phosphatidyl serine movement (flip-flop) can not be demonstrated in PRP as the activated platelets are caught in the emerging clot.

In order to demonstrate flip-flop, we tried to investigate the influence of clots on washed platelets. However, contrary to platelets in a plasma milieu, isolated platelets are damaged by fibrin clots, especially in the presence of thrombin, as can be judged from the appearance of LDH

We conclude that, in PRP, clots induce the appearance of PCP from platelets by vesiculation, possibly accompanied by flip-flop and that thrombin accelerates the process but is not an absolute requirement
High Lipoprotein (a) Levels in Chronic Hemodialysis Patients are Closely Related to the Acute Phase Reaction
Authors: Kario Kazuomi , Matsuo Takefumi , Kobayashi Hiroko , Matsuo Miyako , Asada Reiko , Koide Masanobu
Published: July 2018

To study the mechanism underlying the high lipoprotein (a) [Lp(a)] level in uremic patients on chronic hemodialysis, we investigated the levels of Lp(a), acute phase reactants (C-reactive protein and sialic acid), and interleukin-6 (IL-6) in 54 dialysis patients. The mean [95% Cl] Lp(a) level was increased in the hemodialysis patients compared with the 30 controls (30 [25-36] vs. 18 [14-23] mg/dl, p <0.005). Among dialysis patients, 46% had an Lp(a) level >30 mg/dl, which was significantly higher than the percentage in the control group (17%). The levels of C-reactive protein, sialic acid, and IL-6 were also increased in dialysis subjects compared with controls (200 [134-299] vs. 37 [24-58] μg/dl, p <0.0001; 63 [59-66] vs. 54 [52-56] mg/dl, p <0.002; and 9.2 [7.8-11] vs. 5.5 [5.0-6.1]pg/ml, p <0.0005, respectively). The Lp(a) level was positively correlated with that of C-reactive protein (r = 0.415, p <0.002), sialic acid (r = 0.426, p <0.002), and IL-6 (r = 0.298, p<0.05) in the hemodialysis patients, but not in the controls or non-dialysis uremic patients. The Lp(a) level in the dialysis patients was also positively correlated with activation markers of coagulation (thrombin-antithrombin III complex and plasmin-α2-plasmin inhibitor complex, p <0.005). These results indicate that the Lp(a) level is closely related to the acute phase reaction and hypercoagulability in chronic hemodialysis patients.
Elimination of Acquired Factor VIII Antibodies by Extracorporal Antibody-based Immunoadsorption (Ig-Therasorb®)
Authors: Knöbl P , Derfler K , Korninger L , Kapiotis S , Jäger U , Maier-Dobersberger T , Hörl W , Lechner K , Pabinger I
Published: July 2018

Treatment of potent factor VIII antibodies is a difficult problem. In some cases a reduction ofthe antibody titer is necessary for effective treatment with human factor VIII concentrates. We describe a new method for extracorporal eliminationof factor VIII antibodies (antibody-based immunoadsorption). Blood is drawn from an antecubital vein, citrated, and plasma is separated with a rotating membrane. Plasma passes alternately through one of two columns filled with sepharose-coupled polyclonal sheep antibodies to human immunoglobulins (Ig-Therasorb00), whereas the other column is regenerated. Each cycle has a duration of 15 min. Three patients with high titer factor VIII antibodies (one hemophiliac and 2 with spontaneous antibodies; titers 29, 132, and 313 BU/ml, respectively) were treated. The average reduction of the antibody titer was 76.1 ± 17.2% per session. In each patient 4 sessions were necessary to reduce the antibody titer to < 1 BU/ml. The mean processed plasma volume was 6731 ± 640 ml and the mean duration of each session 3.9 ± 0.7 h. Serum IgG, IgA and IgM levels decreased by 75.3 ± 11.9%, 62.9 ± 19.1%, and 54.8 ± 23.8% respectively. The procedure was tolerated without any side effects. Thus, rapid elimination of factor VIII inhibitors can be achieved with antibody-based immunoadsorption, which can be lifesaving in some cases. This promising method should be evaluated in a larger number of patients.
Factor VIIa in Patients with C1-Inhibitor Deficiency
Authors: Nielsen Erik Waage , Morrissey James , Olsen Jan Ole , Østerud Bjarne
Published: July 2018

In hereditary angioedema (HAE), normal C1-inhibitor (CI-INH) is low and the contact system aetivated. Recently, the findings of a tissue factor mutant selectively deficient in promoting the conversion of FVII to FVIIa, but with retained cofactor for FVIIa, made it possible to examine reliably the pre-existing content of FVIIa in HAH patients. This was of interest as FXIIa (mainly inhibited by (CI-INH) is able to activate FVII directly. FVIIa in 21 remission HAH patients were within normal limits but nearly doubled as compared to their 23 normal siblings (p = 0.0017). Cold promoted activation of FVII (CPA) (common Clot assay) was displayed in plasma of all 5 untreated patients (CI-INH function <35%), but not in plasma of 2 patients treated prophylactically with danazol (CI-INH function about 40%). These results suggest that there is a minute, yet significant activation of FVII in patients with CI-INH deficiency.
In Vivo Recovery of Factor VIII Following Transfusion: A Survey of Recent Data and Publications to Assess the Influence of Standards Used for Potency Assignment
Authors: Prowse C V
Published: July 2018

In the therapy of factor VIII deficiency, experience has taught the value of achieving a target level of plasma coagulation factor VIII. There is a consensus that an empirical value of 2 iu/dl/iu/kg may be used to estimate recovery, and hence plasma levels, of factor VIII. This may be influenced by the methods used to assign label potency to the products and for assessment of patient post-infusion plasmas. To assess a possible influence of the standard used to assign product potency on in vivo recovery, a survey was undertaken of recent in vivo recovery studies. Analysis of submitted data, in combination with published studies over the last ten years, revealed a significant influence of the standard used to assign potency to products on the measured in vivo recovery. Furthermore, from limited data the potency determined in post-infusion patient plasmas was found not to be influenced by the use of one-stage or chromogenic assay methods.
Coagulation Activation and Tissue Necrosis in Meningococcal Septic Shock: Severely Reduced Protein C Levels Predict a High Mortality
Authors: Fijnvandraat Karin , Derkx Bert , Peters Marjolein , Bijlmer Rob , Sturk Augueste , Prins Martin H , Deventer Sander J H van , ten Cate Jan Wouter
Published: July 2018

In 35 consecutively admitted children (mean age: 4.3 years) with a clinical diagnosis of meningococcal septic shock (MSS), activation of the coagulation and fibrinolytic pathways was evaluated directly at admittance to the paediatric intensive care unit (ICU). The association of clinical signs and haemostatic abnormalities was assessed.

All patients had signs of extensive activation of the coagulation system. The 28-day mortality was 26%. Protein C activity was strongly reduced, especially in non-survivors in whom it was significantly lower than in survivors (5% versus 23%; p <0.0001). There was a strong negative correlation between protein C activity and the mean size of the skin lesions (r = -0.71, p <0.001). These results suggest that an acquired protein C deficiency in MSS is related to the pathogenesis of purpura fulminans.

Furthermore, no increase in C4b-binding protein (C4BP) and no decrease in the ratio free protein S/total protein S was observed, suggesting that a deficiency of free protein S as a result of increased C4BP does not play a pathogenetic role in meningococcal septic shock.
Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation
Authors: Bierhaus A , Hemmer Ch J , Mackman N , Kutob R , Ziegler R , Dietrich M , Nawroth P P
Published: July 2018

Serum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.
Coagulation Activation by MC28 Fibrosarcoma Cells Facilitates Lung Tumor Formation
Authors: Francis J L , Amirkhosravi M
Published: July 2018

Tumor cells interact with the hemostatic system in various ways and may thus influence malignant growth and spread. MC28 fibrosarcoma cells possess a potent procoagulant activity (PCA) and form lung tumors following intravenous injection. The aim of this work was to study the relationship between PCA, intravascular coagulation and lung seeding in the MC28 model. MC28 cells were injected into control, warfarinized and heparinized hooded Lister rats. Coagulation changes were monitored by thromboelastography (TEG) and Sonoclot™ analysis (SA), lung fibrin formation by light and electron microscopy, tumor seeding by macroscopic counting and tumor cell and platelet deposition in the lungs by radiolabelling. PCA was measured by chromogenic assay. MC28 PCA was characterized as a tissue factorfactor VIIa complex that probably arose during cell culture or disaggregation of solid tumors. Injection of tumor cells caused marked coagulopathy and was rapidly (within 30 min) followed by fibrin deposition in the lungs and accumulation of radiolabelled platelets. Heparin and warfarin significantly reduced lung seeding (p <0.001) and reduced retention of radiolabelled tumor cells in the pulmonary circulation (p <0.01). Inhibition of cellular PCA by prior treatment with con- canavalin A markedly reduced intravascular coagulation and lung seeding.

We conclude that MC28 cells cause intravascular coagulation as a direct result of their procoagulant activity. The data suggest that tumor cells form complexes with platelets and fibrin which are retained in the lungs long enough for extravasation and seeding to occur.

Anticoagulation reduces tumor cell-platelet-fibrin complex formation, decreasing the time spent in the lungs and thereby reduces seeding. Thus, the antitumor effect of warfarin, at least in the MC28 model, is due to its anticoagulant action.
The Effects of Quality and Timing of Venepuncture on Markers of Blood Coagulation in Healthy Middle-aged Men
Authors: Miller G J , Bauer K A , Barzegar S , Foley A J , Mitchell J P , Cooper J A , Rosenberg R D
Published: July 2018

Effects of the quality and the time of venepuncture on factor VII coagulant activity (VIIC) and the concentrations of fibrinogen, prothrombin fragment 1 + 2 (F1 + 2) and fibrinopeptide A (FPA) were sought in 2665 men, of whom 2334 were re-examined after about one year. Venepunctures were categorised as satisfactory, not fully satisfactory or unsatisfactory according to pre-defined criteria. Neither the quality nor timing of the venepuncture influenced VIICor fibrinogen concentration. However, at baseline and re-examination F1 + 2and FPA were increased on average by about 9% and 45% respectively when venepunctures were not fully satisfactory, and by about 11% and 100% when unsatisfactory. Plasma collected after 1500 h had slightly but significantly lower levels of F1 + 2and FPA than samples taken earlier, possibly due to circadian rhythm. The results emphasise the need for careful surveillance of the venepuncture procedure and the value of FPA when using F1+ 2as a marker of risk of thrombosis.
Contrasting Effects of Thrombin and the Thrombin Receptor Peptide, SFLLRN, on Aggregation and Release of 14C-Serotonin by Human Platelets Pretreated with Chymotrypsin or Serratia marcescens Protease
Authors: Kinlough-Rathbone Raelene L , Perry Dennis W , Packham Marian A
Published: July 2018

Chymotrypsin cleaves glycoprotein Ib (GPIb) on platelets and reduces their responsiveness to thrombin; platelets from patients with the Bernard-Soulier syndrome, which lack GPIb, are also less responsive to thrombin than platelets from normal donors. However, Bernard-Soulier platelets respond normally to the thrombin receptor peptide SFLLRN (13). We compared responses of14C-serotonin-labeled, chymotrypsin-treated platelets (and control platelets) to thrombin (0.25-2 U/ml) and SFLLRN (5–40 μM). Chymotrypsin treatment strongly inhibited thrombin-induced aggregation and release of14C-serotonin when concentrations of thrombin of 0.5 U/ml or lower were used, even though these responses of control platelets remained near the maximum. In contrast, there was little difference between the responses of control and chymotrypsin-treated platelets to SFLLRN, even when the responses of control platelets were less than maximal. Thus, chymotrypsin treatment greatly inhibits the response to thrombin of the seven transmembrane domain thrombin receptor cloned by Coughlin’s group (1, 2). Since Serratia marcescens protease also hydrolyses GPIb, but has less effect than chymotrypsin on other glycoproteins, we pretreated platelets with several concentrations of S. marcescens protease. Concentrations that abolished aggregation and release of14C-serotonin in response to thrombin had little effect on these responses to SFLLRN. One interpretation of these findings would be that by cleaving GPIb, both proteases are affecting an interaction that may be important for activation of the cloned receptor by thrombin, but irrelevant to activation of this receptor by SFLLRN. Alternatively, it may be that in addition to cleaving GPIb, both chymotrypsin and S. marcescens protease also cleave the cloned receptor at a site that results in the loss of SFLLRN, but do not affect the site on this receptor with which SFLLRN interacts.
Axial Dependence of Collagen–Induced Thrombus Formation in Flowing Non-Anticoagulated Human Blood
Authors: Roald Helge E , Sakariassen Kjell S
Published: July 2018

Platelet thrombus formation on collagen fibrils is most pronounced at the upstream end of the surface, and it gradually decreases along the axis in parallel with the direction of the blood flow. This phenomenon, known as axial dependent platelet thrombus formation, is explained by the balance of the platelet supply to the surface and the consumption of platelets by growing thrombi.

In the present study we have affected this balance by (A) inhibiting the growth of platelet thrombi by aspirin (ASA) or clopidogrel, and thus increasing the platelet concentration at the surface, and by (B) utilising blood from cigarette smokers, who have enhanced thrombus formation immediately after smoking, and thus decreasing the platelet concentration at the surface. Thrombus formation in non-anticoagulated blood was triggered by collagen fibrils positioned in a parallel-plate perfusion chamber at a wall shear rate of 2600 s_1which is characteristic for moderately stenosed arteries. Morphometrical assessment of thrombus formation was performed at axial positions of 1 and 13 mm downstream to the blood flow inlet at the collagen surface.

Platelet-collagen adhesion and thrombus volume in blood from nonsmokers were decreased at the downstream location by 39% (p ≤0.0001) and by 60% (p ≤0.0001), respectively. However, increasing the platelet concentration at the surface by partially inhibiting the thrombus growth by ASA or clopidogrel, reduced substantially the axial decrease in platelet adhesion and thrombus volume. The largest reduction was observed with clopidogrel which was also the strongest inhibitor of the thrombus growth at both axial positions investigated. The corresponding figures in blood from smokers with enhanced thrombus formation were 38% (p ≤0.0001) and 72% (p ≤0.001). Thus, enhanced upstream platelet consumption increased the axial reduction in thrombus volume, but not in platelet adhesion.

These data substantiate the view that the “axial dependence phenomenon” may be explained by the balance between the platelet supply to the surface and the consumption of platelets by growing thrombi. It is also apparent that clopidogrel is a more potent inhibitor of platelet thrombus formation than ASA.
Effect of Plasma from Patients with Primary Antiphospholipid Syndrome on Platelet Function in a Collagen Rich Perfusion System
Authors: Reverter Joan-Carles , Tàssies Dolors , Escolar Ginés , Font Josep , Soto Alfonso López , Ingelmo Miguel , Ordinas Antoni
Published: July 2018

The effect on platelet function of plasma from 9 patients with primary antiphospholipid syndrome (PAS) with previous thrombotic episodes was investigated under flow conditions. Five asymptomatic individuals with antiphospholipid antibodies (aPL) (A-aPL) and 14 normal controls were also studied. Patients and controls plasmas were added (1:20 v/v) to anticoagulated blood and perfused through annular chambers containing collagen rich vessel segments. The interaction of platelets with vessel subendothelium was morphometrically evaluated in thin sections. An increase in both covered surface and thrombi formation was observed in perfusions in the presence of PAS-plasma (mean ± SD: 34.2% ± 9.6% and 23.2% ± 10.0% respectively) compared with control plasmas (21.4% ± 7.3% and 10.1% ± 7.7%, p<0.01). Affinity purified anticardiolipin antibodies from one PAS patient showed a similar effect when added to normal blood. In contrast, A-aPL plasma had no effect on platelet-subendothelium interaction. In parallel studies, the same plasmas were incubated with isolated normal platelets before and after activation with ADP or collagen and the binding of immunoglobulins (Ig) was determined by flow cytometry. A significantly increased binding of Ig was observed in 8 out of 9 plasmas from PAS patients when platelets had been activated with collagen but not when resting or ADP activated platelets were used. No increased Ig binding to platelets was seen using A-aPL individuals plasma. These observations might help to explain the pathophysiology of the thrombotic events occurring in patients with PAS.
Systematic Underestimation of Treatment Effects as a Result of Diagnostic Test Inaccuracy: Implications for the Interpretation and Design of Thromboprophylaxis Trials
Authors: Rodgers Anthony , MacMahon Stephen
Published: July 2018

Many clinical trials that have assessed strategies for the prevention of deep vein thrombosis have employed diagnostic tests that are less accurate than venography. The correct interpretation of these trials has been the subject of considerable debate. This paper attempts to quantify the likely effects of the use of inaccurate diagnostic tests (in particular fibrinogen scanning) on the validity and precision of estimates of treatment effects. It is shown that, if there is no difference in the rate of misclassification of deep vein thrombosis between study and control groups, then treatment effects will tend to be systematically underestimated in trials that only use inaccurate diagnostic tests. However, for trials in which there are no false positive diagnoses, such as those employing venography, the estimate of relative treatment effects will be unbiased. This phenomenon is demonstrated empirically by data from randomised trials of antiplatelet therapy. The implications of these findings are two-fold. First, so long as the accuracy of the test is the same in study and control groups, positive trial results cannot be ascribed to diagnostic test inaccuracy. Second, unbiased estimates of relative treatment effects can be obtained by using cheap, practicable and non-invasive screening tests that are supplemented by venographic assessment of positive test results. This would facilitate the conduct of much larger randomised trials than has generally been possible when mandatory venography was used; if future trials randomised some thousands of patients and employed confirmatory venography then they would provide both unbiased and precise estimates of relative treatment effects.
Six Different Point Mutations in Seven Danish Families with Symptomatic Protein C Deficiency
Authors: Lind Bent , Schwartz Marianne , Thorsen Sixtus
Published: July 2018

Six different point mutations of the protein C gene are described in seven Danish families with protein C deficiency associated with an increased risk of venous thromboembolism. All affected family members are heterozygotes for the mutated protein C genotype. One mutation is a G2992→A transition at position +5 in the 5’ splice site of intron D. The other five mutations affect the protein coding region. One is a Cl432→T transition in exon III converting the highly conserved Arg15 to Trp in the Gla-domain. Another mutation is a G3157→C transversion in exon V converting the non-conserved Gly72 to Arg in the epidermal growth factor domain. The remaining three mutations are located in non-conserved amino acid positions in exon IX and affect the serine proteinase domain. The first is a G8559→C transversion converting Gly282 to Arg. The second is a C8571→T transition (present in two families) converting Arg286 to Cys. The third is a C8695→T transition converting Pro327 to Leu. In each family the protein C deficiency cosegregates or probably cosegregates (one family, G8559→C) with the mutation. All affected family members exhibit a reduction of both the antigen and the functional plasma concentration of protein C to approximately 50% of normal indicating that the mutated protein C is not present (type 1 deficiency) or only present in low amounts in plasma. Agarose gel electrophoresis followed by Western blotting shows that the Arg15→Trp substitution is associated with a normal as well as an abnormal migrating plasma protein C band. This provides positive evidence for that both the normal and mutated alleles are expressed (type 2 deficiency). The five other mutations are associated with only one band with the mobility of normal protein C. In one family a novel G1390→A transition converting the normal Ala1 to Thr was demonstrated. This mutation is not linked to the patient specific G8559→C transversion. In conclusion one splice site mutation and five different missense mutations of the protein C gene are described.
Dietary Changes in Fasting Levels of Factor VII Coagulant Activity (FVII: C) Are Accompanied by Changes in Factor VII Protein and other Vitamin K-dependent Proteins
Authors: Bladbjerg E M , Tholstrup T , Marckmann P , Sandström B , Jespersen J
Published: July 2018

The mechanisms behind dietary effects on fasting coagulant activity of factor VII (FVII: C) are not clarified. In the present study of 15 young volunteers, two experimental diets differing in composition of saturated fatty acids (C18:0 [diet S] or C12:0 + C14:0 [diet ML]) were served for 3 weeks each. Fasting blood samples were collected before and after the dietary regimen and analysed for triglycerides, FVII:C, and protein concentrations of FVII, FII, FX, protein C, CRP, albumin, fibrinogen, and F1+2. FVII:C was significantly reduced on diet S compared with diet ML. This was accompanied by a decrease in FVII protein, F1+2 and the vitamin K-dependent proteins FII, FX, and protein C. In contrast, no changes were observed in triglycerides, FVII:C/FVII: Ag, albumin and CRP. Fibrinogen was increased on diet S compared with diet ML. Our findings suggest that the change in fasting FVII:C was part of a general change in concentrations of vitamin K-dependent proteins.
Internalization of an Anti-Glycoprotein IIb/IIIa Antibody by HEL Cells
Authors: Hamamoto Kenjiro , Nomura Shosaku , Suzuki Masahiko , Ohga Shigetoshi , Fukuhara Shirou
Published: July 2018

Platelets are known to internalize monoclonal antibodies directed against the glycoprotein (GP) IIb/IIIa complex. We investigated whether an antibody directed against this complex (NNKY 2-11) was transported from the surface membrane to the intracellular pool in HEL cells. Flow cytometry showed that the percent binding of NNKY 2-11 to the surface membrane of HEL cells was decreased after incubation for 24 h compared with 1 h, while the binding of an anti-GPIb antibody (NNKY 5-5) did not change. It did not seem likely that the GP Ilb/IIIa complex antibody was shed from the surface membrane of the HEL cells during incubation, because the medium conditioned by incubation with these cells for 24 h showed almost no binding to washed platelets. In addition, immunoelectron microscopy demonstrated that GP IIb/IIIa complex antibodies were incorporated into the intracellular pool of HEL cells and were associated with alpha granules. These findings indicated that an anti-GP IIb/IIIa antibody could be internalized by megakaryocytes, as has been previously shown with platelets, suggesting that megakaryocyte GP IIb/IIIa may act as a carrier for various adhesion proteins.
Antithrombin III Concentrates-Are they Clinically Useful?
Authors: Lechner K , Kyrle P A
Published: July 2018

Treatment with AT III concentrates is a good example for the discrepancy between the optimistic expectations based on theoretical considerations or animal experiments and the result of clinical studies. 15 years after the introduction into clinical practice, a benefit for patients treated with AT III concentrates has not been proven. In hereditary antithrombin III deficiency, randomized clinical trials are completely lacking and only few and small sized randomized studies were performed in patients with acquired AT III deficiency. In none of these trials, a significant clinical benefit with regard to reduction of morbidity or mortality was detectable. Based on the published data, one can state that AT III concentrates may be beneficial in some special clinical situations in patients with hereditary antithrombin III deficiency (such as delivery, acute serious thromboembolic complications and postoperative thromboprophylaxis). In acquired AT III deficiency, there is no proven indication for the use of AT III concentrates.
Accelerated Degradation Test of Lyophilized Recombinant Tissue Factor-Liposome Preparations
Authors: van den Besselaar A M H P , Witteveen E , Mansfeld Schaefer-van H , Meeuwisse-Braun J , Strebus A
Published: July 2018

Accelerated degradation experiments were performed to assess the heat stability of lyophilized recombinant tissue factor-liposome preparations from two different manufacturers. When stored at 4° C, these preparations did not show a significant change of the prothrombin time (PT). Two preparations (coded rTF-2 and rTF-a) showed a progressive prolongation of the PT on storage at 30° C, 37° C, and 44° C. The third preparation showed an initial decrease of the PT at 37° C and 44° C followed by a progressive prolongation. Although none of the three preparations was absolutely stable at 30° C, 37° C and 44° C, rTF-2 had the advantage that its PT-ratio (and hence its International Sensitivity Index) did not change under the conditions used in this study. The PT-ratio with a reference material for rabbit brain tissue factor (CRM149S) stored under similar conditions, did not change either.
A Comparative Study of Three Low-molecular Weight Heparins (LMWH) and Unfractionated Heparin (UH) in Healthy Volunteers
Authors: Eriksson Bengt I , Söderberg Karin , Widlund Lars , Wandeli Baback , Tengborn Lilian , Risberg Bo
Published: July 2018

The levels of anti-IIa and anti-Xa activity, as reported in laboratory and clinical studies on low molecular weight heparin (LMWH) preparations, show a high degree of variability. This variation has been proposed as correlated to the variation in incidence of postoperative deep vein thrombosis (DVT) (8-30%) in different LMWH studies on comparable populations undergoing elective hip surgery. The aim of this study was to compare the ex vivo potency of Clexane® (enoxaparin), Fragmin® (dalteparin) and Logiparin® (tinzaparin), applying the concept of bioequivalence, although unknown which activity/activities are best correlated to efficacy. Unfractionated heparin (UH) was included in the study as a reference drug.

The drugs were studied with a cross-over technique in 12 healthy subjects and given subcutaneously in the doses recommended for orthopedic surgery. Blood samples were drawn each hour up to 10 h and at 12 h after administration. Anti-Xa and anti-IIa activities were measured using chromogenic substrate methods

The anti-Xa peak activity (Cmax) and the area under the curve (AUC) were highest for Clexane® and Fragmin® and lower for Logiparin® and UH. Clexane® and Fragmin® were considered bioequivalent in anti-Xa activity. Regarding anti-IIa activity, no bioequivalence was found between the products. Fragmin® was clearly different, with Cmax and AUC approximately twice as high as the other drugs. Whether the demonstrated differences in anti-Xa and anti-II activities are of any clinical significance remains unclear and can only be established by comparative clinical studies.
The Strong Positive Correlation between Factor VII Clotting Activity Using Bovine Thromboplastin and the Activated Factor VII Level
Authors: Kario Kazuomi , Matsuo Takefumi , Asada Reiko , Sakata Toshiyuki , Kato Hisao , Miyata Toshiyuk
Published: July 2018

We compared factor VII clotting activity (FVIIc) assays using different thromboplastins to determine which is the most sensitive for activated FVII (FVIIa) or for FVII antigen (FVIIag). FVIIc levels were measured using thromboplastins derived from bovine brain (FVIIc Bov), human placenta (FVIIc Hum), and rabbit brain (FVIIc Rab). FVIIa levels were measured by fluorogenic assays using human soluble tissue factor (rsTF) or bovine rsTF. We also measured FVII activity by an amidolytic assay (FVIIc:am Hum) using human thromboplastin and a chromogenic substrate for thrombin. FVIIag levels were determined by ELISA. In the FVIIa assay, the reaction time obtained from using bovine rsTF was shorter than that with human rsTF, suggesting that the interaction of plasma FVIIa with bovine rsTF was stronger than with human rsTF. The plasma FVIIa levels measured using human rsTF and bovine rsTF were almost the same (r=0.947, p<0.0001). Among the three FVIIc assays, FVIIc Bov had the strongest positive correlation with the plasma FVIIa level (r=0.886, p<0.000l), but had no correlation with FVIIag. An increase of 1 ng/ml in the plasma FVIIa level yielded a 27.9% increase of FVIIc Bov. Plasma FVIIc Hum and FVIIc:am Hum showed moderate correlations with both FVIIa (r=0.520, p<0.02 and r=0.569, p<0.01, respectively) and FVIIag (r=0.438, p<0.05 and r=0.468, p<0.05, respectively). FVIIc Rab had the lowest correlation with FVIIa (r=0.367, p<0.1), but had a moderate correlation with FVIIag (r=0.436, p<0.05). After in vitro cold activation, FVIIc Bov levels increased the most and FVIIc:am levels showed the least change. These findings indicate that consideration of the thromboplastin used for assay is necessary when assessing the clinical significance of FVII activity as a cardiovascular risk factor.