bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Fibrinolysis after one Year of Treatment with Enalapril in Men and Women with Uncomplicated Myocardial Infarction
Authors: Jansson Jan-Håkan , Nyhlén Katarina , Nilsson Torbjörn , Boman Kurt
Published: March 2018

To study long-term effects of enalapril on mass concentrations of tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI-1), tPA/PAI-1-complex and von Willebrand factor (vWF) in both genders with uncomplicated myocardial infarction.

More than three months after an uncomplicated myocardial infarction 82 survivors (46 males, 36 females) were randomised to enalapril/placebo. PAI-1, tPA, tPA/PAI-1-complex and vWF were measured after two weeks, six and 12 months following randomisation. PAI-1 decreased significantly in both genders in the enalapril-treated group after two weeks, with a maximum decrease at six months (mean reduction: 31% equal to 9.8 µg X L–1, CI: 5.2 to 14.5 µg X L–1, p = 0.0001) and remained significantly lower at 12 months. Mass concentration of tPA decreased significantly (mean reduction; 1.81 µg X L–1, CI: 0.903 to 2.708 µg X L–1, p <0.001) after two weeks treatment in both genders but returned to baseline values at 12 months. The tPA/PAI-1-complex decreased and was significantly lower (mean reduction 0.96 µg X L–1, CI: 0.36 to 1.56 µg X L–1, p = 0.003) in the enalapril group after two weeks and six months (p = 0.037). No decrease of vWF was seen in the enalapril group.

Enalapril treatment up to one year depressed mass concentrations of PAI-1 and transiently tPA and tPA/PAI-1 complex indicating an improvement of the fibrinolytic balance in both genders with uncomplicated myocardial infarction.
Ticlopidine Inhibits the Prothrombotic Effects of Thrombopoietin and Ameliorates Survival after Supralethal Total Body Irradiation
Authors: Gaugler Marie-Hélène , Vandamme Marie , Gourmelon Patrick , Wagemaker Gerard , Van der Meeren Anne , Mouthon Marc-André
Published: March 2018

Thrombopoietin modulates the response of platelets to several agonists and, on the other hand, those agonists can be released following irradiation. Thus, we have determined the effects of thrombopoietin, on its own and in combination with ticlopidine, an anti-platelet drug, on platelet activation, thrombosis formation and survival of irradiated C57BL6/J mice. Administration of thrombopoietin 2 h after 9 Gy total body irradiation increased the 125I-fibrin deposition in mouse tissues and accelerated platelet consumption as revealed by an enhanced drop in platelet counts. Additionally, the number of activated platelets, i.e. those expressing P-selectin on their membrane, was higher in thrombopoietin-treated mice as compared to the placebo group, regardless ex vivo stimulation with agonists. These effects of thrombo-poietin on platelet activation and consumption were reduced when mice were pretreated with ticlopidine. The combination of ticlopidine with thrombopoietin almost fully promoted 180-day survival, reaching the same efficacy as bone marrow transplantation, while only 30% of the mice treated with thrombopoietin alone survived. In summary, thrombopoietin induces long term-mortality of irradiated mice probably through platelet-mediated thrombosis and thus, ticlopidine efficiently counteracts these adverse effects of thrombopoietin.
Effects of Hereditary and Acquired Risk Factors of Venous Thrombosis on a Thrombin Generation-Based APC Resistance Test
Authors: Curvers Joyce , Thomassen M. Christella , Rimmer Janet , Hamulyak Karly , van der Meer Jan , Tans Guido , Preston F. , Rosing Jan
Published: March 2018

Several hereditary and acquired risk factors for venous thromboembolism (VTE) are associated with impaired down-regulation of thrombin formation via the protein C pathway. To identify individuals at risk, functional tests are needed that estimate the risk to develop venous thrombosis.

We determined the effects of hereditary and acquired risk factors of venous thrombosis on an APC resistance test that quantifies the influence of APC on the time integral of thrombin formation (the endogenous thrombin potential, ETP) initiated in plasma via the extrinsic coagulation pathway. APC sensitivity ratios (APCsr) were determined in plasma from carriers of factor VLeiden (n = 56) or prothrombin G20210A (n = 18), of individuals deficient in antithrombin (n = 9), protein C (n = 7) or protein S (n = 14) and of women exposed to acquired risk factors such as hormone replacement therapy (n = 49), oral contraceptive use (n = 126) or pregnancy (n = 35). We also analysed combinations of risk factors (n = 60).

The thrombin generation-based APC resistance test was sensitive for the factor VLeiden and prothrombin G20210A mutation, to protein S deficiency, hormone replacement therapy, oral contraceptive use and pregnancy. The assay was not influenced by antithrombin-or protein C deficiency. The presence of more than one risk factor of venous thrombosis resulted in more pronounced APC resistance. The APCsr of individuals with a single or combined risk factors of VTE correlated well with reported risk increases.

The thrombin generation-based APC resistance test identifies individuals at risk for venous thrombosis due to acquired risk factors and/or hereditary thrombophilic disorders that affect the protein C pathway.
Atorvastatin and Thrombogenicity of the Carotid Atherosclerotic Plaque: the ATROCAP Study
Authors: Cofrancesco Elisabetta , Arbustini Eloisa , Rossi Francesca , Negri Andrea , Tremoli Elena , Gabrielli Livio , Camera Marina , Cortellaro Michele
Published: March 2018

Statins appear to have beneficial effects on fibrous cap stabilisation but their effects on plaque thrombogenicity have not been reported. To evaluate the thrombogenicity of human carotid plaques before and after atorvastatin treatment, 59 patients with bilateral carotid stenosis eligible for two-step carotid endoarterectomy (CEA) were randomly assigned to atorvastatin, 20 mg/day, or placebo. Histological and immunohistochemical analyses, Tissue Factor (TF), Tissue Factor Pathway Inhibitor (TFPI) antigens (Ag) and TF activity were determined in endoarterectomy specimens obtained at baseline and after treatment. Mean TFAg and TFPIAg levels from plaques removed at the first CEA were 55 ± 56 and 32 ± 26 pg/mg. After placebo, TFAg and TFPIAg content was higher in the second than the first CEA. Plaques removed at the second CEA from atorvastatin-treated patients had a lower macrophage content than plaques at the first CEA. TFAg and TFPIAg levels, and TF activity in plaques after atorvastatin treatment were lower (respectively 29, 18% and 56%) than after placebo. These findings indicate that atorvastatin reduce the inflammatory/thrombotic phenotype of carotid plaque, suggesting that these drugs may indeed have a beneficial effect on cerebrovascular events.

We are indebted to Pfizer Italia for providing atorvastatin, 20 mg, and placebo.
Response of Factor VIII and IX-Deficient Blood to Wild Type and High Membrane Affinity Mutant Factor VIIa in an In Vitro Whole Blood Clotting Assay: Possible Correlation to Clinical Outcome
Authors: Henderson Nicole , Key Nigel , Christie Beverly , Kisiel Walter , Foster Donald , Nelsestuen Gary
Published: March 2018

A modified form of the activated whole blood clotting time was used to evaluate response of blood from hemophilia patients to factor VIIa. Repeated assays of individuals over a one-year period showed consistency for each individual and significant difference between individuals. Four hemophilia patients with inhibitors gave low response to factor VIIa in the assay and were characterized as low or moderate clinical responders to factor VIIa therapy. Another four patients with moderate to good clinical responsiveness to factor VIIa therapy showed a strong response to factor VIIa in the assay. A factor VIIa mutant with enhanced membrane affinity showed 10 to 13-fold higher activity than wild type factor VIIa. The results may justify larger studies to determine the utility of this assay for evaluation of patient responsiveness and to set factor VIIa therapy levels.
A Single Thymine Nucleotide Deletion Responsible for Congenital Deficiency of Plasmin Inhibitor
Authors: Yoshinaga Haruhiko , Nakahara Masako , Shibamiya Aya , Nakazawa Fumie , Miles Lindsey , Hirosawa Shinsaku , Aoki Nobuo , Koyama Takatoshi
Published: March 2018

Plasma plasmin inhibitor (PI) is a physiological inhibitor of plasminmediated fibrinolysis and constitutes a hemostatic component in blood plasma; hence its deficiency results in a severe hemorrhagic diathesis. We have carried out molecular analysis of American family members with congenital PI deficiency, and detected a single thymine deletion at nucleotide position 332 in exon 5. The deletion was found in both alleles of the homozygotes and in one allele of the heterozygotes, and the patterns of restriction fragment length polymorphism created by the mutation in the family members were compatible with their phenotypes. The deletion caused a frameshift leading to an alteration and shortening of the deduced amino acid sequence. The amino acid sequence consists of the first 83 amino acids of the N-terminal sequence of the normal PI and additional new amino acids, resulting in a mutant composed of 94 amino acids in contrast to 464 amino acids of the normal PI. In transient expression analysis, the mutant PI whose molecular size was compatible with the predicted amino acid sequence was detected in the lysates of the cells transfected with the mutated PI expression vector. The mutant PI was retained and underwent progressive degradation within the cells, and was minimally excreted into the media. These data indicate that this mutation is the cause of PI deficiency in this pedigree.
Recombinant Soluble P-Selectin Glycoprotein Ligand-Ig (rPSGL-Ig) Attenuates Infarct Size and Myeloperoxidase Activity in a Canine Model of Ischemia-Reperfusion
Authors: Zhou Xiaorong , Zhou Zhongmin , Tarakji Khaldoun , Qin Jian , Sitges Marta , Shiota Takahiro , Forudi Farhad , Schaub Robert , Kumar Anjali , Penn Marc , Topol Eric , Lincoff A. , Wang Kai
Published: March 2018

The role of P-selectin in the process of reperfusion injury was evaluated using a recombinant soluble P-selectin glycoprotein ligand-Ig (rPSGL-Ig) in a canine coronary artery balloon occlusion model. rPSGL-Ig (1 mg/kg) or saline was given as an intravenous bolus 15 min before balloon deflation. Balloon occlusion time was 90 min followed by either 120 min or 7 days reperfusion. Infarct size was significantly reduced in the treatment group when expressed either as percentage of the area at risk or as absolute infarct size. Histological analysis showed that extensive myocardial injury and neutrophil infiltration were reduced by rPSGL-Ig. Myeloperoxidase activity (MPO) was significantly reduced in the risk area in the rPSGL-Ig group. Left ventricular ejection fraction was significantly less impaired during the first 24 h after reperfusion in the rPSGL-Ig group, although there was no difference by 7-day follow-up. Thus, administration of rPSGL-Ig decreases myocardial injury and inflammatory response for at least 7 days after reperfusion of ischemic myocardium.
Measurable Differences between Sequential and Parallel Diagnostic Decision Processes for Determining Stroke Subtype: A Representation of Interacting Pathologies
Authors: Watkins Fred , Jobe Thomas , Helgason Cathy
Published: March 2018

Stroke diagnosis depends on causal subtype. The accepted classification procedure is a succession of diagnostic tests administered in an order based on prior reported frequencies of the subtypes. The first positive test result completely determines diagnosis. An alternative approach tests multiple concomitant diagnostic hypotheses in parallel. This method permits multiple simultaneous pathologies in the patient. These two diagnostic procedures can be compared by novel numeric criteria presented here.

Thrombosis, a type of ischemic stroke, results from interaction between endothelium, blood flow and blood components. We tested for ischemic stroke on thirty patients using both methods. For each patient the procedure produced an assessment of severity as an ordered set of three numbers in the interval [0, 1]. We measured the difference in diagnosis between the sequential and parallel diagnostic algorithms. The computations reveal systematic differences: The sequential procedure tends to under-diagnose and excludes any measure of interaction between pathologic elements.
Effect of Heparin on TAFI-Dependent Inhibition of Fibrinolysis: Relative Importance of TAFIa Generated by Clot-Bound and Fluid Phase Thrombin
Authors: Pentimone Anna , Binetti Bianca , Cramarossa Marialisa , Piro Donatella , Semeraro Nicola , Colucci Mario
Published: March 2018

Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 µg/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through “localized” TAFIa generation.
Transient Platelet Interaction Induces MCP-1 Production by Endothelial Cells via IκB Kinase Complex Activation
Authors: Nothdurfter Caroline , Weber Marion , Fischer Claudia , Ungerer Martin , Gawaz Meinrad , Page Sharon , Massberg Steffen , Brand Korbinian
Published: March 2018

Activated platelets alter endothelial chemotactic and adhesive properties. Transient interaction of α-thrombin-activated platelets with endothelial cells is sufficient to induce secretion of the NF-κBregulated chemokine MCP-1. To evaluate upstream signaling events in platelet-induced NF-κB activation, we compared the effect of platelets, IL-1β or TNF-α on IκB kinase (IKK) complex activation and IκB phosphorylation/proteolysis. Kinase assays demonstrated that platelets induced a slow increase in IKK activity over 30 min, which was similar, but not identical, to IL-1β, whereas TNF-α elicited a rapid induction of IKK. Differential effects were also found on IκB-α/ε degradation, while IKK levels were unaffected. Furthermore, overexpression of kinase-inactive IKK-βKA, as well as NIKKA, inhibited plateletor IL-1β-induced κB-, MCP-1or VCAM-1-dependent transcription. Using adeno-associated virus particles for cell transduction, we found that IKK-βKA substantially reduced stimulus-induced MCP-1 secretion. Platelet-induced signaling and resulting endothelial gene expression may play a role in early atherogenesis as well as plaque progression/destabilization.

Meinrad Gawaz, Sharon Page, and Steffen Massberg contributed equally to this work.
Overexpression of Fibrinogen in ApoE*3-Leiden Transgenic Mice Does not Influence the Progression of Diet-Induced Atherosclerosis
Authors: Rezaee F. , Gijbels M. J. , Offerman E. H. , van der Linden M. , De Maat M. P. M. , Verheijen J. H.
Published: March 2018

Although many epidemiological studies have shown an association between hyperfibrinogenemia and atherosclerosis, it is not established whether elevated fibrinogen has an etiological role in the pathogenesis or is only a reflection of the ongoing disease.

We have studied the contribution of fibrinogen to the development of atherosclerosis in atherosclerosis-prone ApoE*3-Leiden mice that have been cross-bred with transgenic mice overexpressing fibrinogen. Genetic compound offspring were used to evaluate the progression of atherosclerotic lesions after being fed an atherogenic diet for 7 weeks. It was observed that the lesion area of the plaques as well as the severity of the lesions in the aortic valve was comparable in control single transgenic ApoE*3-Leiden mice and in double transgenic apoE*3-Leiden mice overexpressing fibrinogen. No thrombus or fibrin deposition was observed in atherosclerotic lesions in either group of mice.

These results indicate that elevated plasma fibrinogen concentrations in ApoE*3-Leiden transgenic mice do not affect the progression of diet-induced atherosclerotic lesions.
Venous Thrombosis Following the Use of Intermediate Purity FVIII Concentrate to Treat Patients with Von Willebrand’s Disease
Authors: Colvin B. , Gupta V. , Shields M. L. , Smith M. P. , Makris M.
Published: March 2018

We describe four patients with von Willebrand’s disease (VWD) who experienced venous thrombosis after treatment with an intermediate purity factor VIII (FVIII) concentrate (Haemate P®) was used to cover invasive or surgical procedures. Most patients had additional risk factors for venous thromboembolism (VTE) and it is difficult to be certain of the contribution of the concentrate to the VTE. In view of the recognised association between high factor VIII activity (FVIII:C) levels and VTE there is a physiological basis for this complication and it is important to consider this when administering FVIII containing concentrates to VWD patients.
Novel Fibrinogen Truncation with Deletion of Bβ Chain Residues 440-461 causes Hypofibrinogenaemia
Authors: Brennan Stephen , Ockelford Paul , George Peter , Homer Vivienne
Published: March 2018

A 24-year-old male with hepatitis C was initially diagnosed with hypofibrinogenaemia during investigations prior to a liver biopsy. He had a low functional and gravimetric fibrinogen concentration of 1.0 mg/mL and DNA sequencing of all exons, exon-intron boundaries and promoter regions of the fibrinogen Aα, Bβ, and γ genes revealed a single heterozygous g→a mutation at nucleotide 8035 of the Bβ gene. This creates a premature stop at the Trp 440 codon and results in a 22-residue truncation of the Bβ chain. Analysis of purified plasma fibrinogen by SDS PAGE, reverse phase HPLC and ESI MS, however, failed to detect any of the truncated chains in the plasma fibrinogen. The non-expression of aberrant molecules was further confirmed by functional analysis, which revealed normal fibrin polymerisation. The principal structural feature of the independently folding βD domain is its five-stranded anti-parallel β sheet. The deletion here of residues 440 to 461 removes the second strand from this sheet structure and appears to impact on the viability of the nascent chain and its ability to be incorporated into mature fibrinogen molecules. The mutation does not however provoke the formation of hepatic inclusion bodies.
A Reassessment of the Relationship between International Reference Preparations for Human and Rabbit Thromboplastins
Authors: Tripodi A. , van den Besselaar M. H. P. A.
Published: March 2018

Two established international reference preparations (IRP) for thromboplastins, i.e. RBT/90 (rabbit, plain) and rTF/95 (recombinant human, plain) have been calibrated against each other in a 7-centre exercise performed in 2000. The purpose of the study was to compare the calibration results with those of the original calibration study performed in 1995. The international sensitivity index (ISI) for rTF/95 was calculated relative to the ISI for RBT/90. The mean ISI for rTF/95 was 0.961 (between-lab SD 0.028) which was 2% greater than the historical value determined in 1995. There is no indication that the two IRP have deteriorated, but further monitoring of the calibration relationship is recommended.
Mice with a Severe Deficiency of the Endothelial Protein C Receptor Gene Develop, Survive, and Reproduce Normally, and Do not Present with Enhanced Arterial Thrombosis after Challenge
Authors: Liang Zhong , Volkir S. , Haalboom Erijka , Martin J. , Sandoval-Cooper Mayra , Rosen Elliot , Castellino Francis
Published: March 2018

The endothelial cell Protein C receptor (EPCR) functions to enhance activation of anticoagulant Protein C (PC) by the thrombin/ thrombomodulin (Tm) complex on the surface of the endothelium. This overall system functions in anticoagulation, profibrinolytic, and antiinflammatory responses. Mice with a severe targeted deficiency of this receptor have been generated by integration of exogenous DNA elements into the 5’-untranslated region of the EPCR gene. Despite the retention of the entire endogenous EPCR coding sequence in the altered EPCR gene locus, only very low EPCR message contents were detected in mice by quantitative RT-PCR during embryogenesis and up to at least early adulthood. Immunohistochemical analysis of various regions of the arterial tree of mice up to 4 months of age, employing an anti-murine EPCR antibody, confirmed that undetectable levels of this protein were present in arterial regions during these periods. Despite this, these mice are not more prone to arterial thrombosis after challenge in a FeCl3 carotid artery thrombosis model. Small amounts (<10% of wild-type) of this protein were found in other tissues. Matings of mice homozygous for this deficiency led to normal births and survival of the offspring, in contrast to results by others demonstrating early embryonic lethality of a total EPCR deficiency. These data further show that minimal levels of EPCR are able to support male and female virility, as well as embryonic development, birth, and survival to adulthood.
Role of Microthrombus Formation in the Development of Ischemia/Reperfusion-induced Liver Injury in Rats
Authors: Harada Naoaki , Kushimoto Shigeki , Uchiba Mitsuhiro , Okajima Kenji
Published: March 2018

Although tumor necrosis factor-α (TNF-α) has been shown to play a critical role in the pathologic process leading to ischemia/reperfusion (I/R)-induced liver injury in rats by activating neutrophils, it is not clear whether or not microthrombus formation induced by TNF-α contributes to the liver injury. In the present study, we investigated the role of microthrombus formation in I/R-induced liver injury in rats. Hepatic tissue levels of TNF-α were significantly increased after reperfusion, and these were higher in animals subjected to 120 min-hepatic I/R than in those subjected to 60 min-hepatic I/R. Fibrin deposition was observed histologically in the hepatic sinusoidal space only in animals subjected to 120 min-hepatic I/R. Both the decrease in hepatic tissue blood flow and the extent of liver injury in animals subjected to 60 minand 120 min-hepatic I/R were significantly inhibited by pretreatment with anti-rat TNF-α antibody. Although neutrophil elastase inhibitors inhibited the decrease in hepatic tissue blood flow and reduced liver injury in animals subjected to 60 min-hepatic I/R, anticoagulants did not show any effects. Both anticoagulants and neutrophil elastase inhibitors inhibited the decrease in hepatic tissue blood flow and reduced liver injury in animals subjected to 120 min-hepatic I/R. Therapeutic effects of anti-rat TNF-α antibody on the120 min-I/R-induced liver injury were more marked than those of each anticoagulant or each neutrophil elastase inhibitor, and were comparable to those of combined use of anticoagulants and neutrophil elastase inhibitors. These observations strongly suggest that TNF-α induces I/R-induced liver injury primarily by activating neutrophils, and it exacerbates liver injury by inducing microthrombus formation when the production of TNF-α is further increased.
Relationships between Fibrinolytic and Inflammatory Parameters in Human Adipose Tissue: Strong Contribution of TNFα Receptors to PAI-1 Levels
Authors: Bastelica Delphine , Mavri Alenka , Verdier Monique , Berthet Bruno , Juhan-Vague Irène , Alessi Marie-Christine
Published: March 2018

Plasminogen activator inhibitor type 1 (PAI-1), a risk marker of atherosclerosis, is highly expressed in adipose tissue from obese subjects. PAI-1 is also considered as an acute phase protein. Recently, adipose tissue has been described as a source of inflammatory cytokines. Therefore, our aim was to study the relationships between PAI-1, and IL-6, TNF, TNF receptors (TNFRSF1s) and TGFβ1, in plasma and adipose tissue from obese (n = 60) and lean (n = 28) subjects. Study has been extended to plasminogen activators (t-PA and u-PA).

Compared to lean subjects, obese subjects exhibited higher plasma levels of all the studied parameters (except for TGFβ1) whereas in adipose tissue only PAI-1, t-PA and TGFβ1 antigen levels differed. In the obese population, plasma PAI-1 levels were weakly associated with circulating TNF, and this relationship disappeared after adjustment for plasma t-PA. Adipose tissue PAI-1 levels were positively associated with TNFRSF1s and TGFβ1, the strongest relationship being observed with TNFRSF1A, which explained 82% of PAI-1 variability. TNF and IL-6 were the main contributors to t-PA variability in plasma and in adipose tissue, respectively.

Our results argue on the relevance of TNFRSF1s in the regulation of PAI-1 expression by adipose tissue. Association between t-PA, which is mainly produced by endothelial cells, and IL-6 or TNF suggest that inflammation might be involved in angiogenesis in adipose tissue.

Abbreviations: act: activity; ag: antigen; BMI: Body mass index; IL-6: Interleukin-6; PAI-1: plasminogen activator inhibitor type 1; TGFβ1: Transforming Growth Factor beta 1; TNF: Tumor Necrosis Factor alpha; TNFRSF1A and 1B: Tumor Necrosis Factor receptor superfamilly 1A and 1B (TNFRI and II respectively); t-PA: tissue type plasminogen activator; u-PA: urokinase type plasminogen activator
Collagen Response and Glycoprotein VI Function Decline Progressively as Canine Platelets Age in vivo
Authors: Alberio L. , Friese P. , Clemetson K. J. , Dale G. L.
Published: March 2018

Clinical and experimental observations suggest that platelet function deteriorates quickly with cell age. However, efforts to define agedependent alterations have detected only modest biochemical changes occurring late in the cell life span. In this report, we demonstrate two significant alterations of the collagen response occurring during in vivo aging of canine platelets: a progressive increase in the EC50 for collagen types I, III and V and the emergence of a population of aged platelets which are refractory to collagen. Experiments with convulxin, a specific agonist for the collagen receptor glycoprotein VI (GPVI), also demonstrate an age-dependent decline in activation and the appearance of a non-reactive, aged population as observed with native collagens. Our studies indicate that canine platelets have two distinct binding levels for FITC-labeled convulxin and that the higher binding level disappears upon cell aging. During these studies one dog (#428) was identified whose platelets not only failed to demonstrate an age-dependent decrease in convulxin reactivity but also maintained a high convulxinbinding ability throughout their otherwise normal life span. Transfusion of biotinylated platelets from control dogs into dog #428 showed that the expected changes in collagen response and GPVI function did not occur in the transfused platelets. These observations demonstrate that the canine platelet response towards collagen is strongly dependent upon cell-age and suggest that this functional decline is at least partly due to an extrinsic-mediated alteration, possibly proteolytic, of GPVI.

This work was supported in part by grants HL53585 and HL68129 from the National Institutes of Health (GLD) and the Swiss National Science Foundation (SSMBS grant, LA; grant 31-52396.97, KJC). This work has been presented at the XVIII Congress of the International Society on Thrombosis and Haemostasis (Paris, 2001).
Platelet Associated u-PA Up-regulates u-PA Synthesis by Endothelial Cells
Authors: Camoin-Jau L. , Pannell R. , Anfosso F , Bardin N. , Sabatier F. , Sampol J. , Gurewich V. , Dignat-George F.
Published: March 2018

Adhesion of platelets to endothelium has been shown to induce important changes in endothelial properties. In this study, we examined the effect of platelet-endothelial cell interactions on the expression of urokinase-type plasminogen activator (u-PA) by human microvascular endothelial cells. After incubation of endothelial cells with platelets, a dose-dependent increase in the expression of u-PA Ag was observed and reached a plateau for a ratio of 300 platelets per endothelial cells. The u-PA Ag upregulation resulted from an increase in u-PA mRNA that originated from a synthesis by endothelial cells since no u-PA mRNA was detected in platelets. The platelet-induced u-PA synthesis was inhibited when the endothelial cells were pre-treated with phospholipase C to remove the u-PA receptor, or when the platelets were incubated with an antibody that blocks the binding of u-PA to u-PAR. Taken together, these data indicate that u-PA present on the platelet surface interacts with u-PAR on the endothelial cells and induces the u-PA synthesis. This mechanism may represent a physiological control of platelet-mediated intravascular fibrin deposition.
The circulatory half-lives of α -profibrin and α-fibrin monomer, and comparisons with other fibrin(ogen) derivatives
Authors: DiBello Patricia , Shainoff John
Published: March 2018

Previous studies showed that α-fibrin monomer (lacking both A-fibrinopeptides, FPA) is normally cleared from the circulation before it assembles into a clot. Recent studies indicate that substantial quantities of an intermediate, α-profibrin lacking only one of the two FPA are produced in the course of conversion of human fibrinogen to fibrin. Since clearance of the α-fibrin monomer is saturable and receptor mediated, the extent to which α-profibrin or other fibrin(ogen) derivatives might compete for monomer uptake was deemed important. We compared plasma decay of injected human α-fibrin, fibrinogen, and α-profibrin in rabbits using rabbit anti-human fibrinogen for assays. The circulatory half-life of human α-fibrin monomer was short (t1/2 = 2.3 h) and followed a simple exponential decay curve, as anticipated from clearance of rabbit α-fibrin. It was absorbed as fast as it permeated the extravascular space with no redistribution. Human fibrinogen had a long half-life (t1/2 = 39.5 h), calculated from the double exponential plasma decay curves (redistribution + catabolism) observed over 28 h. The α-profibrin had an intermediary half-life (t1/2 = 11 h) determined from double exponential decay curves. Since redistribution accompanied the slow clearance of α-profibrin, its binding by the fibrin receptor(s) must be weak, probably too weak to compete with the clearance of α-fibrin monomer. The initial production of α-fibrin monomer is only partially dependent on prior formation of α-profibrin, as recently shown. Thus, it is the slow clearance and the weak competition from α-profibrin that underlie the occurrence of substantial levels of α-profibrin unaccompanied by detectable levels of α-fibrin monomer in many subjects with vascular disease.
Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative
Authors: Verbeke Koen , Gils Ann , Stassen Jean-Marie , Declerck Paul
Published: March 2018

Interfering with increased levels of plasminogen activator inhibitor-1 (PAI-1) might offer new therapeutic strategies for a variety of cardiovascular diseases. Inactivation of PAI-1 can be accomplished by a number of monoclonal antibodies (MA), including MA-8H9D4. In a previous study, a single-chain variable fragment (scFv-8H9D4) was cloned and found to have the same properties as the parental MA-8H9D4. In the present study, we identified the residues of scFv-8H9D4 that contribute significantly to the paratope. The complementarity determining region 3 from the heavy (H3) and the light (L3) chain were analysed through site-directed mutagenesis. Out of twelve mutations, only four residues appeared to contribute to the paratope. The affinity of scFv-8H9D4-H3-L97D for PAI-1 was 38-fold decreased (KA = 4.8 ± 0.2 × 107 M–1 vs. 1.8 ± 0.7 × 109 M–1 for scFv-8H9D4) whereas scFv-8H9D4-H3-R98Y did not bind to PAI-1. The affinities of scFv-8H9D4-L3-Y91S and scFv-8H9D4-L3-F94D for PAI-1 were 9- and 5-fold reduced, respectively, whereas the combined mutation resulted in an 86-fold decreased affinity (KA = 2.1 ± 0.2 × 107 M–1).In accordance with the affinity data, these mutants had no, or a reduced, PAI-1 inhibitory capacity, confirming that these four particular residues form the major interaction site of scFv-8H9D4 with PAI-1. In combination with the three-dimensional structure, these data contribute to the rational design of PAI-1 neutralizing compounds.
Stabilization of monocyte chemoattractant protein-1-mRNA by activated protein C
Authors: Marx Antje , Weiler Hans , Liebe Volker , Lang Siegfried , Kaden Jens , Zieger Wolfgang , Borggrefe Martin , Huhle Guenter , Haase Karl , Brueckmann Martina
Published: March 2018

The activated protein C (APC) pathway has been suggested to be a common link between coagulation and inflammation. APC may function to restore hemostasis via modulation of cytokine expression. We investigated the effect of APC on the endothelial expression of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is controlled by the activation of central proinflammatory transcription factors, such as nuclear factor-kappa B (NF-κ B).

We found that human APC (2.5-10 μ g/ml) upregulated the amount of MCP-1-mRNA in human umbilical vein endothelial cells (HUVEC) and caused a time- and dose-dependent increase in MCP-1 protein production (p <0.001 for APC 2.5 μg/ ml at 4 up to 24 h). In this cell culture model MCP-1 induced an improvement of cell migration and wound repair after injury to endothelial monolayers. After stimulation of MCP-1-mRNA-transcription with TNF-α (0.1-1 ng/ml), HUVEC’s were washed and an inhibitor of gene transcription, Actinomycin D (1 μg/ml), was added in the presence or absence of APC. HUVEC’s receiving APC contained more MCP-1-mRNA than controls after one hour and up to eight hours suggesting an inhibitory effect of APC on MCP-1-mRNA degradation (with APC: 753 ± 56 atto mol of MCP-1-mRNA per ml of cell lysate vs. 263 ± 60 atto mol/ml without APC at t =4 h; p <0.001). Electrophoretic mobility shift assays revealed that APC attenuated NF- κB DNA-binding capacity implying that NF- B may not be involved in the upregulatory effect of APC on MCP-1 production.

The ability of APC to upregulate the production of MCP-1, most likely by increasing the stability of MCP-1-mRNA rather than by transcriptional activation via NF- B, identifies a novel immunomodulatory pathway, by which APC may control the local inflammatory reaction, thereby initiating wound repair and modulating the extent of endothelial injury.
Vascular endothelial growth factor enhances venous thrombus recanalisation and organisation
Authors: Waltham Matthew , Burnand Kevin , Collins Michael , McGuinness Catharine , Singh Inderjit , Smith Alberto
Published: March 2018

Vascular endothelial growth factor (VEGF) is a regulator of physiological and pathological angiogenesis and is found in naturally resolving experimental venous thrombi, where it may also regulate recanalisation. In this study VEGF protein was injected into venous thrombi to determine if this enhanced recanalisation and organisation. A rat model of inferior vena cava (IVC) thrombosis was used. Thrombi were formed in 3 groups (n = 3 per group). 10 μl 125I-VEGF was directly injected into thrombus thirty minutes after induction. Three hours, 1 day and 6 days later thrombus, IVC, and other tissues were harvested. 125I-VEGF was mostly distributed in the thrombus and the IVC, with smaller amounts in other tissues. Thrombi were formed in a further 4 groups (n = 6 per group).Thirty minutes after induction control solution or 1 ng, 10 ng or 100 ng recombinant human VEGF165 was injected directly into the thrombus. Lumen recanalisation, thrombus organisation and monocyte content were measured on digitised sections by image analysis. In animals treated with 10 ng of VEGF there was a greater area of lumen recanalisation [mean 5492 pixels, standard error of mean (sem) 922] compared to controls (mean 2974, sem 385) (P = 0.005). There was a significant increase in the organisation score in all treated animals (1 ng: mean 70, sem 1.7, P = 0.0025; 10 ng: mean 70, sem 2.0, P = 0.0042; 100 ng: mean 72, sem 1.9, P = 0.0003) compared to controls (mean 63, sem 1.7). The monocyte content was lower in the animals treated with 1 ng VEGF (mean 3.8% of thrombus area, sem 0.3%) compared to controls (mean 5.5%, sem 0.4%) (P = 0.0008).The proportion of monocytes migrating to the centre of the thrombus increased in a dose-related manner. VEGF may prove to be of use in the treatment of venous thrombosis.
Impaired Procoagulant-anticoagulant Balance during Hormone Replacement Therapy?
Authors: van Baal W. , Emeis Jef , van der Mooren Marius J. , Kessel Hilda , Kenemans Peter , Stehouwer Coen
Published: March 2018

In this randomised, placebo-controlled 12-week study, sixty healthy postmenopausal women received either placebo (N = 16) or daily 2 mg micronised oestradiol, either unopposed (N = 16, E2 group) or combined with a progestagen for 14 days of each cycle (N = 28, E2+P group).

As compared to placebo, plasma levels of AT III were reduced only in the E2 group (∼28%), plasma levels of protein C decreased only in the E2+P group (∼4%) and plasma levels of protein S decreased in both the E2 and E2+P group (∼21%). In both the E2 and E2+P groups, the plasma levels of factor VII (antigen and activity) showed a borderline significant increase (∼10%), whereas no significant change was observed in active factor VII. Plasma levels of tissue-type plasminogen activator (∼22%), urokinase plasminogen activator (∼25%) and plasminogen activator inhibitor type-1 (∼43%) decreased in the E2 and E2+P groups, whereas those of plasminogen increased (∼12%). Treatment was associated with an increase in levels of prothrombin fragment 1+2 (∼31%), but levels of thrombin-antithrombin III complexes, and of plasmin-α2-antiplasmin complexes and total fibrin(ogen) degradation products did not change significantly.

Short-term E2 and E2+P treatment is associated with a shift in the procoagulant-anticoagulant balance towards a procoagulant state. A substantial proportion of women do not have a net increase in fibrinolytic activity. These data may be relevant in explaining the increased risk of venous thromboembolism associated with ERT and HRT, and possibly also in explaining the negative results of the Heart and Estrogen/progestin Replacement Study.
Rescue of Fatal Neonatal Hemorrhage in Factor V Deficient Mice by Low Level Transgene Expression
Authors: Yang Tony , Cui Jisong , Taylor Jeremy , Yang Angela , Gruber Stephen , Ginsburg David
Published: March 2018

Factor V (FV) is a critical component of the coagulation cascade. FV-deficient patients suffer moderate to severe bleeding, though residual FV activity is detectable in nearly all cases. In contrast, FVdeficient mice die either during mid-embryogenesis, or of massive perinatal hemorrhage. In order to examine the requirements for FV in murine embryogenesis and hemostasis, we generated transgenic mouse lines expressing a Fv minigene under control of either the tissue-specific albumin (Malb) or rat platelet factor 4 (Rpf4) promoter. A total of 12 Malb and 3 Rpf4 lines were analyzed. Though expression in the target tissue was detectable in most lines by RT-PCR, only low levels of transgene expression were achieved (<3% of endogenous Fv in all lines). Despite a low level of Fv transgene expression, rescue of the lethal Fv
−/− phenotype was observed with one of the Malb transgenic (Tg+) lines. However, rescue appeared to be incomplete with continued loss of >1/2 of expected Tg+, Fv
−/− mice in early embryogenesis. Rescued Tg+, Fv
−/− mice have undetectable FV (<0.1%) in both plasma and platelet compartments, but survive the perinatal period and mature to adulthood without spontaneous hemorrhage. We conclude that FV present at <0.1% is sufficient to support postnatal survival. Failure of the Malb transgene to rescue the midembryonic block suggests that FV expression is required during mammalian development at higher levels or with a different tissue-specific or temporal pattern. Taken together, these data may explain the observation of residual FV activity in most human FV-deficient patients due to early embryonic lethality in those absolutely deficient, and suggest that minimal levels of FV expression, below the level of detection, also may be sufficient to support survival in humans.
Collagen Binding Assay for von Willebrand Factor (VWF:CBA): Detection of von Willebrands Disease (VWD), and Discrimination of VWD Subtypes, Depends on Collagen Source
Authors: Favaloro Emmanuel
Published: March 2018

A large number of different collagen preparations [n = 21] have been assessed for their ability to both detect von Willebrands Disease (VWD), and discriminate different VWD subtypes. Collagen preparations were tested at a range of concentrations and included: Type I, III and IV, and various mixtures of these, as aqueous supplied preparations and/or reconstituted from bulk lyophilised stock. Tissue sources for collagens ranged from human placenta to calf skin to equine tendon. Three of the collagen preparations tested did not support von Willebrand factor (VWF) binding in an ELISA process (therefore unable to detect VWD). The ability of the remaining preparations to detect VWF was variable, as was their ability to discriminate VWD subtypes. Detection of VWF and discrimination of VWD subtypes was not mutually inclusive. Thus, some collagen preparations provided excellent detection systems for VWF, but comparatively poorer discrimination of Type 2 VWD, while others provided good to acceptable detection and discrimination. Subtype discrimination was also dependent on the collagen concentration, and some batch to batch variation was evident with some preparations (particularly Type I collagens). Overall, best discrimination was typically achieved with Type I/III collagen mixtures, or Type III collagen preparations (where effectiveness was highly dependent on concentration). Good discrimination was also achieved with a commercial Type III collagen based VWF:CBA kit method. Results of the various ‘VWF:CBA assays’ are also compared with those using the Ristocetin Cofactor (VWF:RCof) assay (by platelet agglutination) and that using a commercial ‘VWF:RCof-alternative/ activity’ ELISA procedure. These latter methodologies tended to be less sensitive to VWF-discordance when compared to that detected by the majority of the VWF:CBA procedures.

Abbreviations: FVIII:C Factor VIII: coagulant (assay); HMW High Molecular Weight [VWF]; PNP Pooled Normal Plasma; RIPA Ristocetin induced platelet aggregation procedure; VWD von Willebrands disease; VWF von Willebrand Factor; VWF:Ag von Willebrand Factor Antigen (assay); VWF: CBA Collagen Binding [Activity] Assay for VWF; VWF:RCof Ristocetin Cofactor Assay for VWF
Von Willebrand Disease Type 2M “Vicenza” in Italian and German Patients: Identification of the First Candidate Mutation (G3864A; R1205H) in 8 Families
Authors: Federici Augusto , Budde Ulrich , Castaman Giancarlo , Drewke Elke , Krey Sonja , Mannucci Pier , Riesen Gabi , Rodeghiero Francesco , Zieger Barbara , Zimmermann Rainer , Schneppenheim Reinhard
Published: March 2018

Von Willebrand disease type 2M “Vicenza” (VWD 2M V) is characterised by autosomal dominant inheritance, low von Willebrand factor (VWF) and the presence of “supranormal” multimers in plasma. This specific phenotype has been described in Italian and recently also in German patients. The molecular defect is linked to the VWF gene. However, no specific mutations have been identified until now. We analysed the complete coding region and adjacent intron sequences of the VWF gene in Italian families in comparison to German families with VWD 2M V by a PCR-based mutation screening, combined with SSC-and heteroduplex-analysis of exons 2 through 52, followed by direct sequencing. We identified the first heterozygous candidate mutation (G3864A; R1205H) in all affected members of the 7 Italian families and in 1 German patient but not in the unaffected family members nor on 100 chromosomes of normal subjects, suggesting a causal relationship between the mutation and the phenotype. Haplotype identity, with minor deviations in one Italian family, suggests a common but not very recent genetic origin of R1205H.
Nucleotide Sequence of the Gene Encoding Murine Tissue Factor Pathway Inhibitor-2
Authors: Kazama Yoshiaki , Kamei Shintaro , Kuijper Joseph , Foster Donald , Kisiel Walter
Published: March 2018

Summary

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a 32 kDa extracellular matrix-associated serine proteinase inhibitor consisting of three tandemly-arranged Kunitz-type domains. Two overlapping genomic clones containing sequences encoding murine TFPI-2 were isolated from a A FIXII 129 SVJ mouse genomic library, and the complete nucleotide sequence of the gene was determined. The murine TFPI-2 gene spans approximately 9.3 kilobases and consists of five exons and four introns. The nucleotide sequences surrounding all the exon-intron boundaries are highly conserved and obey the GT-AG rule. Each Kunitz-type domain is encoded by a single exon, similar to that observed for other Kunitz-type proteinase inhibitors. A total of 1,577 bp of the 3’-flanking region contains a probable polyadenylation site (ATTAAA) at +5,759 and an apparent cleavage or termination site (CATTG) at +6,170. The 5’-flanking region of the murine TFPI-2 gene contains a prototypical TATA box, a GC box and two CAAT boxes. In addition, several candidate transcription factor binding sites responsible for placenta-, endothelial cell-, and smooth muscle cell-specific expression of the TFPI-2 gene were also identified.