bluuebunny/crossref_metadata_2025 huggingface dataset:
The Reciprocal Influences of Working Memory and Linguistic Knowledge on Language Performance: Considerations for the Assessment of Children With Developmental Language Disorder
Authors: Archibald Lisa M. D.
Published: July 2018

Purpose

This article considers how the language performance of school-age children with language impairments, such as developmental language disorder, is influenced by the reciprocal relationship of existing linguistic knowledge and working memory resources and the resultant implications for assessment.

Method

A viewpoint is provided by reviewing working memory theory, empirical evidence of the reciprocal relationship between working memory and existing language knowledge, and critically evaluating available standardized and nonstandardized tools designed to assess working memory or linguistic skills.

Conclusions

Speech-language pathologists with an excellent understanding of the reciprocal relationship between working memory and linguistic knowledge will need to examine performance across tasks and contexts varying in these demands in order to achieve an accurate clinical profile of relevant strengths and weaknesses for individual children.
Math Difficulties and Working Memory Growth in English Language Learner Children: Does Bilingual Proficiency Play a Significant Role?
Authors: Swanson H. Lee , Kong Jennifer , Petcu Stefania
Published: July 2018

Purpose

The purpose of this study was to determine those components of working memory (WM) that play a significant role in predicting math growth in children who are English language learners (
N
= 157) with serious math difficulties (MD).

Method

A battery of tests was administered in English and Spanish that assessed computation, reading, vocabulary, inhibition, and components of WM in Grade 1 children with follow-up testing in Grades 2 and 3.

Results

The results indicated that growth in the executive component of WM was related to growth in math performance. Proficient bilingual children (proficient in both Spanish and English vocabulary) with MD outperformed less proficient bilingual children with MD on measures of math calculation, fluid intelligence, reading, and Spanish WM at Grade 3.

Conclusion

Growth in the executive component of WM is significantly related to growth in math computation, and increased bilingual proficiency across testing waves yielded positive gains in both math and cognitive performance in children with MD.
Pull the Andon Rope on Working Memory Capacity Interventions Until We Know More
Authors: Gillam Sandra , Holbrook Sarai , Mecham Jamie , Weller Daylene
Published: July 2018

Purpose

The purpose of this article is to discuss the current state of interventions for improving working memory (WM) capacity language and academic skills and to provide suggestions for speech-language pathologists working with students who have WM capacity limitations.

Method

Meta-analyses, systematic reviews, randomized controlled trials, and nonrandomized comparison studies investigating the role of WM interventions for improving WM capacity language and academic skills are reviewed. Strategies for improving WM are discussed.

Results

The use of interventions designed to improve WM capacity and other cognitive skills is currently not supported by the research. Direct WM interventions should be considered to be experimental at this time. Such interventions require further investigation before they are used regularly for children with developmental language disorders.

Discussion

Clinicians and practitioners should look to already established interventions for improving how students with developmental language disorders utilize organizational strategies and other well-researched methods for improving their cognitive and academic functioning in functional contexts.
A New Assay Based on Thrombin Generation Inhibition to Detect Both Protein C and Protein S Deficiencies in Plasma
Authors: Duchemin Jérôme , Pittet Jean-Louis , Tartary Michel , Béguin Suzette , Gaussem Pascale , Alhenc-Gelas Martine , Aiach Martine
Published: July 2018

Activated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.
Inhibition of the Metastasis of Lewis Lung Carcinoma by Antibody Against Urokinase-Type Plasminogen Activator in the Experimental and Spontaneous Metastasis Model
Authors: Kobayashi Hiroshi , Gotoh Junko , Shinohara Hiromitsu , Moniwa Nobuhiko , Terao Toshihiko
Published: July 2018

A selective inhibitory antibody, raised against human high molecular weight urokinase-type plasminogen activator (HMW-uPA), was examined to determine whether it would inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. Polyclonal antibody to human uPA cross-reads with the murine uPA and inhibits murine uPA activity. When examined with an in vitro assay system using a modified Boyden chamber, the anti-catalytic IgG to uPA suppressed the invasion of tumor cells through Matrigel. Anti-uPA IgG inhibited neither the cell proliferation nor the binding of tumor cells to Matrigel, and showed no significant suppression of chemotactic migration of tumor cells to tibronectin. In an in vivo spontaneous metastasis assay, multiple subcutaneous (s. c.) injections of anti-uPA IgG (up to a concentration of 200 pg [ 500 inhibitory unit/mousc/day]) for 7 days immediately after s. c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of anti-uPA IgG. In an in vivo experimental metastasis assay, multiple s. c. injections of anti-uPA IgG for 7 days after intravenous (i. v.) tumor cell inoculation did not reduce the number of lung tumor colonies. These results suggest that uPA more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation, during the metastatic process.
Defibrotide Stimulates Expression of Thrombomodulin in Human Endothelial Cells
Authors: Zhou Quansheng , Chu Xiaohong , Ruan Changgeng
Published: July 2018

Cultured human umbilical vein endothelial cells were incubated with defibrotide at concentrations of 0, 5, 50 and 500 jxg/ml for 4 and 24 h respectively. Thrombomodulin activity and molecules on the surface of the cells were determined by chromogenic assay and radioimmunoassay, thrombomodulin antigen in endothelial cells and in conditioned medium of the cells was measured by immunoradioassay. Thrombomodulin mRNA within the cells was analysed by slot blot. After 24 h of incubation, the activity and molecules of thrombomodulin on the surface of endothelial cells, as well as the antigen and mRNA of thrombomodulin in the cells were significantly increased in a dose dependent manner. However, the level of thrombomodulin antigen in conditioned medium was about equal to that of the control. Our data indicate that defibrotide stimulates expression of thrombomodulin in human endothelial cells. These beneficial effects may play a role in antithrombotic activity of defibrotide.
Fibrinogen Plasma Levels as a Marker of Thrombin Activation: New Insights on the Role of Fibrinogen as a Cardiovascular Risk Factor
Authors: Ceriello Antonio , Pirisi Mario , Giacomello Roberta , Stel Giuliana , Falleti Edmondo , Motz Enrico , Lizzio Sebastiano , Gonano Fabio , Bartoli Ettore
Published: July 2018

Fibrinogen has recently emerged as a major risk factor for athero- thrombosis. However, the pathophysiologic mechanism linking high fibrinogen concentration to cardiovascular disease is unclear.

In this study 136 subjects (75 males, 61 females, age 51.7 ± 14.4 years, mean ± standard deviation, range 17-82) were tested for total and HDL-cholesterol, total triglycerides, apolipoprotein AI, apolipo- protein B, fibrinogen, prothrombin fragment and D-dimer. Moreover, 5 subjects who had hyperfibrinogenemia (range 450 to 950 mg/dl) for at least 6 months by repeated measurements, were treated with a short 7-day course of heparin 12,500 U/day subcutaneously. The effect of heparin on all the above mentioned parameters was observed at the end of treatment and after 7 days of wash-out. Simple regression analysis detected a positive correlation between fibrinogen and age, prothrombin fragment and D-dimer, and a negative correlation between fibrinogen and HDL-cholesterol and apolipoprotein AI. A direct correlation between age and both prothrombin fragment and D-dimer was also demonstrated. Multivariate analysis showed a persistent correlation between fibrinogen and prothrombin fragment, D-dimer and age, that was not influenced by sex, smoking habit and body mass index. In the 5 hyperfibrinogenemic subjects, heparin administration significantly reduced fibrinogen (625.4 ± 211.1 vs 455.2 ± 112.3 mg/dl, p <0.03), prothrombin fragment (0.97 ± 0.1 vs 0.63 ± 0.2 nM, p <0.002) and D-dimer (336 ± 101.8 vs 275.2 ± 78.5 ng/ml, p <0.03). All these parameters returned to baseline after 7 days of wash-out (fibrinogen 632.5 ± 198.2 mg/dl, prothrombin fragment 0.96 ± 0.2 nM, D-dimer 325.8 ±98.65 ng/ml).

These data indicate that fibrinogen plasma levels are strongly correlated to parameters of thrombin activation in plasma and suggest that high fibrinogen plasma levels might be a risk marker for cardiovascular disease because expression of an existing thrombophilia.
Reversible Inhibition of Human Platelet Activation by Hypothermia In Vivo and In Vitro
Authors: Michelson Alan D , MacGregor Hollace , Barnard Marc R , Kestin Anita S , Rohrer Michael J , Robert Valeri C
Published: July 2018

A hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22° C and 37° C. the forearm skin temperature was maintained at temperatures between 22° C and 37° C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB 2 (the stable metabolite of TXA 2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37° C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB 2
ggeneration, and the bleeding time. In summary, by a combination of immunologic, biochemical, and functional assays, we demonstrate that hypothermia inhibits human platelet activation in whole blood in vitro and in vivo. Rewarming hypothermic blood completely reverses the activation defect. These results suggest that maintaining normothermia or rewarming a hypothermic bleeding patient may reduce the need for platelet transfusions.
Inherited Diseases of Platelet Glycoproteins: Considerations for Rapid Molecular Characterization
Authors: Bray Paul F
Published: July 2018

The characterization of inherited diseases of platelets has provided valuable information about platelet physiology and platelet protein function. Genetic studies on patients with Glanzmann thrombasthenia, the Bemard-Soulier syndrome, and platelet-type von Willebrand disease have been confined to abnormalities of the GPIIb-IIIa and GPIb-IX receptor complexes. The primary molecular technique used in these analyses has been the polymerase chain reaction (PCR). The amplified PCR products are either directly sequenced, or used to screen for abnormal regions of the genes which are then sequenced. This review examines the known mutations in GPIIb-IIIa and GPIb-IX, focusing on those genetic issues which should dictate decisions regarding the approach to identifying molecular defects. The techniques for characterizing mutant alleles in Glanzmann thrombasthenia and Bernard-Soulier syndrome are described and a general strategy is offered. Because mutations resulting in reduced levels of transcripts can be missed when screening RNA, an argument is made for using genomic DNA as the primary material for mutation detection.
Upper Extremity Impedance Plethysmography in Patients with Venous Access Devices
Authors: Horne McDonald K , Jo Mayo Donna , Alexander Richard , Steinhaus Elizabeth P , Chang Richard C , Whitman Eric , Gralnick Harvey R
Published: July 2018

Central venous access devices (VADs) are often associated with thrombotic obstruction of the axillary-subclavian venous system. To explore the accuracy of impedance plethysmography (IPG) in identifying this complication we performed IPG on 35 adult cancer patients before their VADs were placed and approximately 6 weeks later. At the time of the second IPG the patients also underwent contrast venography of the axillary-subclavian system. The venograms revealed partial venous obstruction in 12 patients (34%) and complete obstruction in two (5.7%). Although the IPG results from venographically normal and abnormal patients overlapped extensively, mean measurements of venous outflow were significantly lower in the patient population with abnormal venograms (P = 0.052 for Vo; P = 0.0036 for Vo/Vc). In our hands, therefore, upper extremity IPG cannot be used to make clinical decisions about individual patients with VADs, but it can distinguish venographically normal and abnormal populations.
The Interaction of β2 Glycoprotein-I and Heparin and Its Effect on β2 Glycoprotein-I Antiphospholipid Antibody Cofactor Function in Plasma
Authors: McNally T , Cotterell S E , Mackie I J , Isenberg D A , Machin S J
Published: July 2018

β2 glycoprotein-I (β2GPI), a cofactor for antiphospholipid antibody (aPA) binding, binds to many anionic macromolecules including heparin. The nature of this interaction with heparin is not well understood and its effect on the purported biological functions of β2GPI is unknown.

We have examined the interactions of dermatan sulphate (DS) and different pharmaceutical preparations of heparin with β2GPI by crossed immunoelectrophoresis (CIE) and investigated the effect of these agents on plasma levels of p2GPI antigen (β2GPI: Ag) by a standardised enzyme linked immunosorbent assay (ELISA). P2GPI aPA cofactor activity (β2GPI:Cof) was also measured using a modified solid phase an-ti-phosphatidylserine (aPS) ELISA. CIE results confirmed a heparin-β2GPI interaction with unfractionated (UF) heparin. β2GPI:Ag levels were unaffected by any of the preparations investigated. There were no significant differences in β2GPI:Cof activities of the samples containing LMW heparins or DS but levels of β2GPI:Cof were increased in samples containing UF sodium and calcium heparin preparations (0.5 IU/ml Monoparin, p <0.05, and 10 IU/ml Liquemin and Calcipa-rine, p <0.05).
Release of Tissue-Type Plasminogen Activator in Response to Muscarinic Receptor Stimulation in Human Forearm
Authors: Jern Sverker , Selin Lena , Bergbrant Anders , Jern Christina
Published: July 2018

We have recently shown that mental stress increases local net release of tissue-type plasminogen activator (t-PA) across the forearm vascular bed. However, the mechanisms responsible for the t-PA release in man during stress are undefined. To study the effects of endothelial cell receptor stimulation and fluid shear stress we used the perfused forearm model to characterize the in vivo tissue plasminogen activator (t-PA) response in man to methacholine (Mch) and sodium nitroprus-side (SNP), at doses calculated to cause similar degrees of vasodilation. The study was performed in 7 healthy young men (age 22-24 yrs) without hypertension, diabetes mellitus, or hypercholesterolemia. Each subject received double-blind step-wise i. a. infusions of Mch (0.1-0.8-4.0 μg/min) and SNP (0.5-2.5-10 μg/min) in randomized order. Each dose step was infused for 5 min. Forearm blood flow was assessed by plethysmography. Net release/uptake was expressed as the product of arterio-venous concentration gradient and forearm plasma flow. At pre-infusion baseline, there was a significant net release of t-PA antigen of approximately 0.9 ng × min−1 × 100 ml−1 and t-PA activity of 3.5 fmol × min−1 × 100 ml−1 across the forearm. I. a. infusion of Mch and SNP increased forearm blood flow from 1.9 to 14.9 and from 1.8 to 12.1 ml × min−1 × 100 mb1, respectively (Mch vs SBP N. S.). Net release of t-PA antigen increased more than 10-fold from 0.9 to 10.7 ng × min−1 × 100 ml−1 after Mch, but fell after SNP (2-way . ANOVA, Mch vs SNP p = 0.05). t-PA activity net release increased from 4.0 to 100 fmol × min−1 × 100 ml−1 after Mch, but switched to a net uptake after SNP (Mch vs SNP p = 0.02). No consistent changes of PAI−1 antigen were observed during either infusion. It is concluded, that endothelial muscarinic receptor stimulation causes a marked increase in net release of t-PA antigen and activity, in conjunction with endothelium-dependent vasodilation. By contrast, increased fluid shear stress per se by receptor-independent vasodilation has little effect on t-PA release.
The Tyrosine Kinase Inhibitors, Genistein and Methyl 2,5-Dihydroxycinnamate, Inhibit the Release of (3H)Arachidonate from Human Platelets Stimulated by Thrombin or Collagen
Authors: Hargreaves Philip G , Licking Ellen F , Sargeant Paul , Sage Stewart O , Barnes Michael J , Farndale Richard W
Published: July 2018

We have investigated the effects of the tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, on [3H]arachidonic acid release from human platelets. Both tyrosine kinase inhibitors blocked, in a dose-dependent manner, the release of arachidonic acid stimulated by thrombin or suspensions of collagen fibres. Blockade by the tyrosine kinase inhibitors occurred early in the arachidonate release time course. Both genistein and methyl 2,5-dihydroxycinnamate also inhibited tyrosine phosphorylation in platelets. The inhibitors were specific in that they did not affect protein kinase C activity, ATP levels or mobilization of Ca2+ from internal stores. These findings suggest a role for tyrosine kinase activity in the regulation of phospholipase A2 in platelets stimulated by the physiological ligands, thrombin and collagen
The Effect of Subcutaneous Injection of Unfractionated and Low Molecular Weight Heparin on Thrombin Generation in Platelet Rich Plasma - A Study in Human Volunteers
Authors: Bendetowicz A Vicky , Kai Hu , Knebel Richard , Caplain H , Hemker H Coenraad , Lindhout Theo , Béguin Suzette
Published: July 2018

We administered a dose of unfractionated heparin (UFH) and two doses of a low molecular weight heparin (LMWH) to healthy volunteers by SC injection. The doses given were: a) UFH, 5000IU, which represents 8.7 mg of >5,400 MW active heparin (ACLM) and no <5,400 active heparin (BCLM), b) enoxaparin 40 mg (3.4 mg ACLM, 2.2 mg BCLM) and c) enoxaparin 1 mg/kg body weight (on the mean 75 mg, containing 6.4 mg ACLM and 4.1 mg BCLM). We determined the effect on thrombin generation in platelet rich plasma (PRP) between 1 and 8 h after injection. UFH administration caused only a 5-8% inhibition of the thrombin potential (i. e. the area under the thrombin generation curve). Significantly higher inhibition of the thrombin potential was seen after administration of both doses of enoxaparin. To wit 9-26% at the low dose and 29-46% at the high dose. UFH injection caused a prolongation of the lag-time before the thrombin burst. Only with the high dose of enoxaparin the lag-times were significantly more prolonged with enoxaparin than with UFH.

Excess amounts of platelet factor 4 (PF4) were able to neutralize completely the anti-thrombin activity in normal plasma spiked with enoxaparin as well as in plasma samples obtained after SC enoxaparin injection. With a large excess of PF4 the anti-factor Xa activity could be inhibited to a maximum of 50%. This indicates that ACLM (above critical length material, MW >5400) is neutralized completely by PF4 whereas BCLM (below critical length material, MW <5400) is not. The anti-thrombin heparin-activity, hence the ACLM fraction of heparin, was shown to have disappeared from the serum of PRP samples. The BCLM fraction was found after coagulation of PRP in concentrations that were indistinguishable from those in the PPP.

We conclude that in PRP the activity of the BCLM fraction of injected LMWH remains after platelet activation. The possible role of this activity in thrombin inhibition and in the antithrombotic action of low molecular weight heparins is discussed.
The Influence of Fibrinogen and Fibrin on Thrombin Generation - Evidence for Feedback Activation of the Clotting System by Clot Bound Thrombin
Authors: Kumar Rachana , Béguin Suzette , Hemker H Coenraad
Published: July 2018

In plasma the bulk of thrombin generation takes place after a clot has formed. We therefore investigated in what way the clot influences thrombin generation in plasma. The forming clot withdraws thrombin from free solution. Consequently less thrombin activity is found and less thrombin-inhibitor complexes are formed. The thrombin that is adsorbed to the clot reduces the lag time before thrombin generation in intrinsically or extrinsically triggered platelet poor plasma as well as in platelet rich plasma. We investigated the mechanism of this activation.

Clots were obtained by recalcification of plasma or by the addition of thrombin-like enzymes (Reptilase, Agihal) from snake venoms. They were thoroughly washed until the washing fluid was devoid of any detectable clotting enzyme activity. In platelet poor plasma (PPP), thrombin-induced clots shorten the factor Va-dependent lag-time of thrombin generation in the extrinsic system as well as the factor VUIa-dependent thrombin generation in the intrinsic system. Factor V or factor VII preparations that in itself hardly influence thrombin generation patterns aquire the capacity to shorten these lag-times when incubated with clot. The last washing fluid of the clot is inactive. Snake venom induced clots are not active either. Clots that are incubated in heparinised plasma for 1 h or more are as active as clots from normal plasma are. A role of factor Xa can not be excluded but must be minor because a clot made by addition of thrombin to plasma from which the factors II, VII, IX and X have been removed is as active as a clot from normal plasma is.

When added to recalcified platelet rich plasma (PRP), in which the lag-time of thrombin formation is dependent upon activation of platelet procoagulant phospholipid activity, any type of clot shortens the lagtime before the burst of thrombin generation. Clots that are obtained by snake venom enzymes are also active in this system. This indicates that fibrin alone is capable to induce the procoagulant phospholipid activity in platelets.

We conclude that three known thrombin-dependent feedback activations in the clotting system (factor V, factor VIII and platelets) are efficiently supported by thrombin bound to the fibrin clot and that there is an additional activating effect of fibrin on the procoagulant action of platelets.
Interaction of Antistasin-Related Peptides with Factor Xa: Identification of a Core Inhibitory Sequence
Authors: Ohta Noriya , Brush Michael , Jacobs John W
Published: July 2018

Antistasin, isolated from the Mexican leech, is a 119 amino acid protein which is a selective and potent inhibitor of coagulation Factor Xa. Previous studies indicated that an arginine residue located at position 34 of the inhibitor was cleaved by Factor Xa during the inhibition reaction. To evaluate this residue as the reactive site of antistasin, and to define shorter fragments of antistasin displaying Factor Xa-inhibitory activity, a series of peptides were synthesized corresponding to amino acids 27- 49 of the inhibitor. The most potent peptide synthesized was a disulfide-bridged, 19 amino acid peptide, ATS29-47, which inhibited Factor Xa with a Ki = 35 nM, and increased plasma clotting times by over 4-fold at a concentration of 33 uM. Reduction or sulfation of the cysteine residues in ATS29-47 reduced Factor Xa inhibitory activity by over 95%. Peptides as short as seven residues corresponding to position 33-39 of antistasin displayed Factor Xa inhibitory activity. The peptides did not inhibit thrombin or trypsin at concentrations 1000-fold higher than used in Factor Xa assays. The shortest peptide displaying anticoagulant activity in human plasma was the disulfide-bridged peptide, D-Arg-Cys-Arg-Val-His-Cys-Pro, which increased clotting times by 50% at micromolar concentrations. These results demonstrate that antistasin-related peptide sequences can serve as model structures for the development of novel, low molecular weight anticoagulants.
Effects of a Low Molecular Weight Heparin (Fragmin®) and of Unfractionated Heparin on Coagulation Activation at the Site of Plug Formation In Vivo
Authors: Elchinger Sabine , Wolzt Michael , Nieszpaur-Los Malgorzata , Schneider Barbara , Lechner Klaus , Eichler Hans-Georg , Kyrle Paul A
Published: July 2018

The clinical benefits of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) have been shown in many trials. However, the mode of action of heparin has not been fully elucidated. Thus, we wanted to study the effects of UFH and LMWH in vivo by measuring coagulation activation markers in blood obtained directly from a vascular injury site. In a double-blind, randomized, 3-way, cross-over study 18 healthy volunteers were given UFH (150 U/kg s.c.) and 2 doses of LMWH [35 U/kg s.c. (low dose, Id), 75 U/kg s.c. (high dose, hd)]. Prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and fibrinopeptide A (FPA) were measured in bleeding time blood and in venous blood before and after drug application. In addition, the effects of UFH and LMWH on in vitro coagulation tests were studied. Compared to base line, UFH and both IdLMWH and hdLMWH caused significant reductions of F1+2, TAT and FPA in bleeding time blood at 2 h. A marked effect of UFH and of hdLMWH was also seen at 5 h. The inhibition of FPA generation was more pronounced after hdLMWH compared to IdLMWH. In venous blood, UFH and LMWH caused reductions of F1+2, but not of TAT and FPA. In vitro, UFH predominantly affected the anti-IIa assays (activated partial thromboplastin time, thrombin time) and LMWH mainly the anti-Xa test system. Using a technique that investigates the activated coagulation system in vivo, a time- and dose dependent inhibitory effect of heparin on coagulation activation was detectable. Therefore, in our experimental setting a preferential inhibition of a particular portion of the coagulation system by one of the two heparin preparations was not detectable.
In Vitro Mutagenesis Study of Two Critical Glutamic Acids in the Calcium Binding Loop of the Factor IX Heavy Chain
Authors: Hamaguchi Nobuko , Stafford Darrel
Published: July 2018

We investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium’s absence, factor IXGlu235Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245LyS’s conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium. We conclude that glutamic acids 235 and 245 are, as predicted, involved in the calcium binding site in the factor IX catalytic domain, and that the correct conformation of this calcium binding loop is essential for maintaining the active site pocket.
Possible Structural Implications of 20 Mutations in the Protein C Protease Domain
Authors: Greengard Judith S , Griffin John H , Fisher Cindy L
Published: July 2018

Analysis of naturally occurring protein mutations yields valuable insights into functionally important sequences. Characterizing mutations responsible for protein C deficiency at the molecular level has been the subject of intensive investigation. In a previous study, a three-dimensional model of the serine protease domain of protein C was used to analyze the set of protease domain mutations previously available. The mutations were largely found to fall into a limited number of categories. A recently updated protein C mutation data base has provided a number of new mutations which have been analyzed for structural predictions.
“Spontaneous” Platelet Aggregation in Whole Blood in Diabetic and Non Diabetic Survivors of Acute Myocardial Infarction
Authors: Gray Rosaire P , Hendra Timothy J , Patterson David L H , Yudkin John S
Published: July 2018

There is increasing evidence that platelet thrombi play an important role in the pathogenesis of acute myocardial infarction (AMI). We compared “spontaneous” platelet aggregation in whole blood in 17 non-diabetic and 12 diabetic subjects on admission with AML There was no significant difference in the fall in platelet count between the two groups, expressed as platelets remaining (75.2 ± 7.9% vs 77.3 ± 6.9% at 10 min, 66.6 ± 8.9% vs 68.5 ± 6.3% at 20 min, 63.5 ± 8.2% vs 64.9 ± 6.7% at 30 min and 59.4 ± 10.3% vs 61.3 ± 7.6% at 60 min). The rate of “spontaneous” aggregation was increased in subjects with evidence of heart failure on admission compared to those without (59.9 ± 7.9% vs 66.2 ± 6.6% at 30 min [p = 0.05] and 55.4 ± 9.6% vs 63.1 ± 7.7% at 60 min [p = 0.04]). There was no correlation between the fall in platelet count and admission plasma glucose, glycated heaemoglobin or peak aspartate aminotransferase. The subjects studied on admission with AMI had greater rates of “spontaneous” aggregation than 8 subjects studied between 6 and 12 months after acute myocardial infarction (75.9 ± 7.4% vs 85.8 ± 5.4% at 10 min; p = 0.001 and 64.3 ± 7.5% vs 75.0 ± 7.8% at 30 min; p = 0.006) and compared to normal controls (90.7 ± 4.4% at 10 min; p <0.001 and 83.4 ± 6.5 at 30 min; p <0.001). This study provides evidence of increased “spontaneous” platelet aggregation in subjects admitted with acute myocardial infarction but no difference between diabetic and non-diabetic subjects was observed.
Protamine Neutralization of the Release of Tissue Factor Pathway Inhibitor Activity by Heparins
Authors: Harenberg Job , Siegele Marietta , Dempfle Carl-Erik , Stehle Gerd , Heene Dieter L
Published: July 2018

The present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa- activity were taken, in order to detect a possible relation between the remaining anti-factor X a activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs.

There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamine

It is assumed that protamine displaces heparins from the binding sites of TFPI. There were no differences between UF- and LMW-heparin. The data indicate that the sustained antifactor Xa activity after antagonization of LMW-heparins as well as heparin rebound phenomena are not mediated by TFPI activity.
Inhibition of Integrin-Mediated Platelet Aggregation, Fibrinogen-Binding, and Interactions with Extracellular Matrix by Nonpeptidic Mimetics of Arg-Gly-Asp
Authors: Varon David , Lider Ofer , Dardik Rima , Shenkman Boris , Alon Ronen , Hershkoviz Rami , Kapustina Galina , Savion Naftali , Martinowitz Uri , Greenspoon Noam
Published: July 2018

The interaction of the activated platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) with fibrinogen and von-Wille-brand factor (vWF) is essential for platelet aggregation. The minimal structure required for this integrin’s binding to fibrinogen is the Arg-Gly-Asp (RGD) sequence. Inasmuch as normal level of GPIIb-IIIa-RGD interactions are required for maintaining hemostasis, elevated platelet aggregation can cause adverse pathological effects. We have previously reported that nonpeptidic mimetics of RGD, consisting of carboxylate and guanidinium groups of Asp and Arg divided by a linear 11-atom spacer, acquired a significant affinity for the GPIIb-IIIa integrin and inhibited platelet aggregation. The structural requirements for the interactions of the RGD sequence with GPIIb-IIIa and the inhibitory potential of a newly designed series of mimetics on platelet aggregation and interactions with extracellular matrix (ECM) were assayed herein. Adenosine-diphosphate (ADP)-induced platelet aggregation was inhibited in a dose-dependent manner by various RGD mimetics, with a maximal inhibition of 80-100% with an IC50 of 3 μM for the most potent inhibitor, NS-11 which a six-membered ring was introduced into the spacer chain, which exceeded the IC50 attained with the original RGDS peptide. The inhibitory effect of the RGD mimetics was attributed to their specific interaction with the GPIIb-IIIa integrin, since these mimetics inhibited the binding of the PAC-1 mAb to GPIIb-IIIA. Furthermore, the binding of 125I-labeled fibrinogen to platelets was inhibited by the RGD surrogates in a dose-dependent and saturable manner. The RGD-mimetics also inhibited up to 70% the adhesion, aggregation, and deposition of platelets onto ECM. Thus, we suggest that the novel nonpeptidic mimetics of RGD described herein, which were shown to be resistant to proteolytic digestion, would be valuable in novel therapeutic approaches to treat in RGD-dependent pathological disorders involving platelet-ECM interactions.
Effects of Homocysteine and Related Compounds on Prostacyclin Production by Cultured Human Vascular Endothelial Cells
Authors: Wang Jian , Dudman Nicholas P B , Wilcken David E L
Published: July 2018

Elevated plasma homocysteine is associated with an increased risk of intravascular thrombosis. Platelet aggregation and thrombosis are inhibited by prostacyclin produced by the vascular endothelium. Our aim was to investigate whether homocysteine and related metabolites inhibit endothelial prostacyclin production. We used a radioimmunoassay for 6-ketoprostaglandin-F1α to assay medium which had been in contact with confluent cultured endothelial cells. In medium containing 20% human serum, endothelial prostacyclin production was not specifically inhibited by homocysteine, S-adenosylhomocysteine or protein-bound homocysteine. Further, there was no consistent difference in prostacyclin production by cells cultured in medium containing sera from homocystinuria patients, compared with medium containing normal healthy sera. We conclude that vascular disorder in homocystinuria is unlikely to result from effects of homocysteine or related metabolites on endothelial prostacyclin production. By contrast, S-adenosylhomocysteine and protein-bound homocysteine specifically inhibited prostacyclin production by cells cultured in medium containing 20% fetal calf serum.
The Influence of Insulin, ß-Estradiol, Dexamethasone and Thyroid Hormone on the Secretion of Coagulant and Anticoagulant Proteins by HepG2 Cells
Authors: Niessen René W L M , Pfaffendorf Birgit A , Sturk Augueste , Lamping Roy J , Schaap Marianne C L , Hack C Erik , Peters Marjolein
Published: July 2018

As a basis for regulatory studies on the influence of hormones on (anti)coagulant protein production by hepatocytes, we examined the amounts of the plasma proteins antithrombin III (AT III), protein C, protein S, factor II, factor X, fibrinogen, and prealbumin produced by the hepatoma cell line HepG2, into the culture medium, in the absence and presence of insulin, β-estradiol, dexamethasone and thyroid hormone. Without hormones these cells produced large amounts of fibrinogen (2,452 ± 501 ng/mg cell protein), AT III (447 ± 16 ng/mg cell protein) and factor II (464 ± 31 ng/mg cell protein) and only small amounts of protein C (50 ± 7 ng/mg cell protein) and factor X (55 ± 5 ng/mg cell protein). Thyroid hormone had a slight but significant effect on the enrichment in the culture medium of the anticoagulant protein AT III (1.34-fold) but not on protein C (0.96-fold) and protein S (0.91-fold). This hormone also significantly increased the amounts of the coagulant proteins factor II (1.28-fold), factor X (1.45-fold) and fibrinogen (2.17-fold). Insulin had an overall stimulating effect on the amounts of all the proteins that were investigated. Neither dexamethasone nor ß-estradiol administration did substantially change the amounts of these proteins.

We conclude that the HepG2 cell is a useful tool to study the hormonal regulation of the production of (anti)coagulant proteins. We studied the overall process of protein production, i.e., the amounts of proteins produced into the culture medium. Detailed studies have to be performed to establish the specific hormonal effects on the underlying processes, e.g., transcription, translation, cellular processing and transport, and secretion.
Transcriptional Regulation of Tissue- and Urokinase-type Plasminogen Activator Genes by Thrombin in Human Fetal Lung Fibroblasts
Authors: Hayakawa Yumiko , Tazawa Shigeki , Ishikawa Tai-ichi , Niiya Kenji , Sakuragawa Nobuo
Published: July 2018

The mechanism of thrombin induction of tissue- and urokinase-type plasminogen activator (t-PA and u-PA) biosynthesis was investigated in cultured human fetal lung fibroblast cells, IMR-90. Northern blot analysis of total RNA from thrombin-treated cells showed marked accumulations of both t-PA and u-PA mRNA during 24 h. Nuclear run-on experiments showed that the transcription rates of both genes were increased in the thrombin-treated cells.

These thrombin effects were inhibited by cycloheximide (CHX), an inhibitor of protein biosynthesis. Treatment of IMR-90 cells with CHX alone caused an increase in u-PA mRNA but not in t-PA mRNA. CHX, however, did not affect the transcription rates of both genes in the cells. Thus, on-going protein synthesis is required for increased accumulations of both t-PA and u-PA mRNA by thrombin but not for the constitutive expression of u-PA gene in IMR-90 cells. Therefore, we conclude that the accumulations of t-PA and u-PA mRNA due to thrombin result mainly from increased rates of their gene transcriptions, and that this influence is exerted in part by proteins synthesized by thrombin stimulation.

Thrombin also increased plasminogen activator inhibitor type-1 (PAI-1) in the levels of both antigen and mRNA more rapidly than it increased t-PA in IMR-90 cells. In conditioned medium, most of the secreted PAI-1 seemed to form a complex with t-PA. Northern blot analysis using a PAI-2 cDNA probe showed that the levels of PAI-2 mRNA were markedly increased in response to thrombin. PAI-2 appeared not to have been secreted by the cells because the antigen was rarely detectable in the conditioned medium. Thus, thrombin increases not only plasminogen activators, but their inhibitors as well.
Relationship between Endothelial Tissue Factor and Thrombogenesis under Blood Flow Conditions
Authors: Diquélou Armelle , Dupouy Dominique , Gaspin Dominique , Constans Jacques , Sié Pierre , Boneu Bernard , Sakariassen Kjell S , Cadroy Yves
Published: July 2018

We have evaluated the relationship between the level of tissue factor (TF) expression by stimulated endothelial cells and thrombus formation under blood flow conditions. Cultures of human umbilical venous endothelial cells (HUVECs) were treated in order to express different levels of TF activity. They were stimulated for 4 h with either I) lipopolysaccharides (LPS, 10 µg/ml), II) recombinant interleukin Iß (IL1ß, 50 Ul/ml) or III) simultaneously with LPS and IL1ß (LPS + IL1ß). TF activity was low on confluent HUVECs or on the corresponding extracellular-matrix (ECM prepared by exposure of HUVECs to 0.1 N NH4OH). In contrast, it was high when HUVECs were stimulated with LPS or IL1ß, and significantly higher (p <0.05) with LPS+IL1ß. The TF activity associated with the stimulated ECM was 2-fold higher (p <0.05) than that expressed on the luminal surface of the stimulated HUVECs, irrespective of the agonist or combination of agonists used.

These surfaces were exposed to non-anticoagulated human blood at a venous (50 s-1) and an arterial (650 s-1) wall shear rate in parallel-plate perfusion chambers for 5 min. Thrombus formation was morphologically quantified by measuring the deposition of platelets and fibrin. Fibrin deposition was also immunologically quantified. Fibrin deposition was related to the level of TF expression. Non-stimulated HUVECs and corresponding ECMs were not thrombogenic. The luminal surface of HUVECs stimulated with LPS or IL1ß alone expressed low levels of TF activity and was a poor inducer of platelet deposition and fibrin deposition (<15%) at 50 s-1. In contrast, fibrin deposition increased to 80% when the cells were stimulated with LPS and IL1ß simultaneously. This fibrin deposition was comparable to that found on the corresponding ECM, despite a two-fold lower TF activity. However, at 650 s-1, platelet and fibrin deposition on HUVECs stimulated with LPS + IL1ß were significantly lower than that observed on the corresponding ECM. In all circumstances, the thrombogenicity was TF-dependent, since fibrin deposition was totally blocked by anti-TF antibodies. Thus, it appears that the level of TF activity expressed on endothelial cells governs thrombus formation. However, the impact of TF expression on thrombus formation is also affected by the blood flow.
The Reproductive Health of Women with von Willebrand Disease Unresponsive to DDAVP: Results of an International Survey
Authors: Foster Paul A
Published: July 2018

An international registry was established on the reproductive health of women with types of von Willebrand disease (vWd) unresponsive to DDAVP. Data was collected on 44 women from 16 treatment centers in nine countries. Severe menorrhagia requiring blood product therapy occurred at least once in 80% of the women for whom data was reported. Most of the reported episodes occurred prior to the diagnosis of vWd and/or the use of oral contraceptive (OC) therapy. OC therapy was clinically effective in the treatment of chronic menorrhagia in 22 of 25 (88%) women treated. Two of the women, however, were unable to tolerate chronic OC use and a third became refractory to treatment. Hysterectomy was performed in 10 of the 44 women (23%). The reported indication for six of the procedures was menorrhagia. Seventy percent were performed at two treatment centers, suggesting different thresholds for the performance of the procedure. There were 69 pregnancies reported in 31 of the 44 women. Fifteen of the pregnancies resulted in spontaneous abortions. The incidence of miscarriage was 22% and appeared clustered, with 10 of 15 of the miscarriages occurring in just four of the women