bluuebunny/crossref_metadata_2025 huggingface dataset:
Inconsistency of Association between Type 1 von Willebrand Disease Phenotype and Genotype in Families Identified in an Epidemiological Investigation
Authors: Eikenboom Jeroen , Bertina Rogier , Rodeghiero Francesco , Castaman Giancarlo
Published: March 2018

In a previous epidemiological investigation among schoolchildren of Northern Italy, a conservative 1% prevalence of type 1 von Willebrand disease (VWD) was found. Diagnosis was based on a positive family history and low von Willebrand factor (VWF) ristocetin cofactor activity. To investigate whether the type 1 VWD phenotype as detected by our original methodology cosegregates with one or more specific alleles of the VWF gene, we have performed genotype analysis in affected subjects and their family members.

Eleven of the 14 subjects previously identified as having VWD, all with mild personal bleeding symptoms, agreed to participate in the genetic study. Remarkably, the laboratory measurements of the previous investigation were completely confirmed in 10 of the 11 subjects. Clear cosegregation of the VWD type 1 and a specific VWF allele was observed in one family and was likely in the family of two other pro-bands. In three additional propositi and their families a possible association of the phenotype with a VWF allele was found. No association was observed in the remaining five subjects and their families. During 13-year follow-up few additional bleeding episodes were recorded among investigated subjects, most often occurring in the one family manifesting clear cosegregation.

The results of this study illustrate that a personal and family bleeding history and persistently low VWF ristocetin cofactor activity, fitting the usual criteria for type 1 VWD, may not cosegregate with genetic markers at the VWF gene locus. Thus the prevalence of VWD defined as a disorder involving the VWF locus might be overestimated in population study. However, phenotypic diagnosis still remains fundamental to identify patients at risk of bleeding. Further research should clarify whether in families with more severe clinical and laboratory phenotype a clear association with markers of VWF is found.
Inhibition of Thrombin-induced Neuronal Cell Death by Recombinant Thrombomodulin and E5510, a Synthetic Thrombin Receptor Signaling Inhibitor
Authors: Sarker Krishna Pada , Abeyama Kazuhiro , Nishi Jun-ichiro , Nakata Masanori , Tokioka Takeshi , Nakajima Toshihiro , Kitajima Isao , Maruyama Ikuro
Published: March 2018

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a pivotal role in thrombosis and haemostasis. Thrombin acts as a mitogen and apoptosis inducer in a dose-dependent fashion. We have previously shown that thrombin caused proliferation of vascular smooth muscle cells (VSMCs). Here, we show that a low concentration of thrombin caused proliferation of mouse neuroblastoma (Neuro-2a) and human neuroblastoma (NB-1) cells, while higher concentrations affected cell viability in a time-dependent manner. Similar effects were observed when thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) was applied. The dying cells showed nuclear condensation and fragmentation, suggesting that cell death occurred by apoptosis. The extent to which thrombin induced cell death was significantly attenuated by recombinant thrombomodulin (rTM), or by a minimum functional domain of TM, termed E456. Furthermore, a synthetic compound that inhibits signaling from the thrombin receptor, 4-cyano-5,5-bis (4-methoxyphenyl)-4-pentanoic acid (E5510), and the antioxidant N-acetyl L-cysteine (NAC), efficiently prevented thrombin-induced Neuro-2a cell death. Thus, thrombin inhibitors and antioxidant appear to neutralize thrombin toxicity.
Hormonal State rather than Age Influences Cut-off Values of Protein S: Reevaluation of the Thrombotic Risk Associated with Protein S Deficiency
Authors: Liberti G. , Bertina R. M. , Rosendaal F. R.
Published: March 2018

Of the well known risk factors for thrombosis protein S deficiency is one of the most difficult to diagnose with certainty. Reliable estimates for the prevalence of protein S deficiency in the general population are not available and the risk of thrombosis is a controversial issue. It has been shown that levels of protein S fluctuate over time. However the determinants of low levels of protein S in the healthy population are not clear. Therefore, we evaluated the influence of sex, age and hormonal state on the antigen levels of protein S in 474 healthy control subjects of the Leiden Thrombophilia Study (LETS). In univariate analysis, sex, age, oral contraceptive (OC) use and post-menopausal state all influenced protein S antigen levels. In a multivariate model for the whole sample only menopausal state and OC use had still an effect on the levels of total protein S and only menopausal state had an independent effect on the values of free protein S. On the basis of this analysis we established different cut-off levels for these subgroups and we re-evaluated in the Leiden Thrombophilia Study the risk of thrombosis for individuals with low protein S using these different reference ranges. With these specific cut-off points, we did not observe an increase in the risk of thrombosis in patients deficient of total protein S (OR 1.2, 95% CI 0.5-2.9) or free protein S (OR 1.3, 95% CI 0.5-3.5). When men and women were analyzed separately, the risk in women was 1.5 (95% CI 0.4-5.4) and 2.4 (95% CI 0.6-9.2) for total and free protein S deficiencies, respectively; and there was no increase in thrombotic risk for men. We conclude that it may be helpful to apply separate cut-off levels in the assessment of protein S levels. This does not, however explain the differences between our results and those of others in the estimate of thrombotic risk of protein S deficiency.
Hyperhomocysteinemia, Atherosclerosis and Thrombosis
Authors: Cattaneo Marco
Published: March 2018

High plasma levels of homocysteine are the results of the interplay between congenital and environmental factors. In the last two decades, a growing amount of interest has focused on mild-to-moderate hyperhomocysteinemia as a risk factor of thromboembolic diseases. Case-control and cross-sectional studies clearly indicated that mild-to-moderate hyperhomocysteinemia is associated with heightened risk of both arterial and venous thrombosis. On the other hand, prospective studies did not unequivocally show that hyperhomocysteinemia is associated with a high thrombotic risk. Therefore, additional studies are needed to define whether hyperhomocysteinemia is a risk factor for thrombosis, especially of the venous circulation. Among these, prospective cohort studies will clarify better the temporal relationship between high homocysteine levels and the thrombotic event. Most importantly, however, randomized, placebo-controlled, double-blind trials of the effects of homocysteine-lowering vitamins on the thrombotic risk are urgently needed. Not only will they help in defining whether the relationship between hyperhomocysteinemia and thrombosis is causal, they will also have a potential dramatic impact in the prevention of thromboembolic events.
Inhibitor Antibody Development and T Cell Response to Human Factor VIII in Murine Hemophilia A
Authors: Qian Jiahua , Borovok Marina , Bi Lei , Kazazian Haig , Hoyer Leon
Published: March 2018

In order to understand better the mechanism of inhibitor formation in hemophilia A patients, we have characterized the immune response to human factor VIII in a murine model of hemophilia A. Mice with severe factor VIII deficiency caused by targeted gene disruptions in exons 16 and 17 were injected intravenously with human factor VIII. Anti-factor VIII was absent or was detected at only very low levels in hemophilic mice of both strains after a single injection of 0.2 μg factor VIII, but it was present in most mice after a second exposure. Subsequent exposures led to high titer anti-factor VIII antibodies in both ELISA and inhibitor assays. A human factor VIII-specific T cell proliferative response was detected with spleen cells obtained three days after a single injection with human factor VIII, before mice had detectable anti-factor VIII antibodies. Subsequent exposures to factor VIII were followed by an increased T cell proliferative response. These studies indicate that murine hemophilia A is a good model for the study of the immune response to human factor VIII, especially the role of the T cell in the early steps in inhibitor antibody formation.
Effects of Diets Enriched in Lauric, Palmitic or Oleic Acids on Blood Coagulation and Fibrinolysis
Authors: Temme Elisabeth , Hornstra Gerard , Mensink Ronald
Published: March 2018

In this study we compared the effects of specific saturated fatty acids (lauric acid and palmitic acid) with those of a monounsaturated fatty acid (oleic acid) on coagulation and fibrinolytic parameters in healthy women and men. Eighteen women and fourteen men consumed, in random order, three experimental diets, each for six weeks. The diets consisted of solid foods and approximately 70% [28 percent of energy (En%)] of the fat calories was supplied. As determined from duplicate portions, in the lauric acid diet 7.3 En% and in the palmitic acid diet 6.1 En% of oleic acid were exchanged for lauric or palmitic acid, respectively. The lauric acid diet also contained some (average 1.8 En%) more myristic acid.

Compared with the oleic acid diet, factor VIIam in the female sub jects was 9% higher with the lauric acid diet (P = 0.0036; 95% CI, 3 to 14%) and 10% higher with the palmitic acid diet (P = 0.0011; 95% CI, 5 to 16%). Changes in men were not significant.

Plasminogen Activator Inhibitor (PAI-1) activity was higher on the palmitic acid compared with the oleic acid diet (difference between diets of 2.3 U/ml; P = 0.0098; 95% CI, 0.4 to 4.3 U/ml) and the lauric acid diet (difference between diets of 2.2 U/ml; P = 0.0123; 95% CI, 0.2 to 4.1 U/ml).

No significant differences between diets were observed for antithrombin III activity, fibrinogen concentrations, fragment 1+2 concentrations, plasminogen or α2-antiplasmin activity.

From this study, we conclude that diets rich in lauric or palmitic acid, compared with a diet rich in oleic acid, unfavourably influence factor VIIam activity, in a gender specific manner. In addition, the plasminogen activator inhibiting capacity of the plasma is impaired with a palmitic acid rich diet compared with an oleic or lauric acid rich diet.
A 96-well Microfiltration Assay for Measurement of Total Clottable Fibrinogen
Authors: Keller A. , Argirion S. , Heene D. L. , Dempfle C. E.
Published: March 2018

In clinical routine use, fibrinogen is measured by clotting-time methods, or by clot turbidity in photometric prothrombin time determination. For calibration of these assays measurement of total thrombinclottable protein has been recommended. We have now developed a microfiltration assay for total thrombin-clottable protein. Plasma samples were mixed with thrombin in a 96-well microfiltration device. After clot formation, the fluid was extracted by vacuum suction, and fibrin adherent to the filter membranes washed with buffer. Membrane segments with adherent fibrin were recovered from the 96-well manifold with a punch and transferred to tubes containing denaturing buffer solution. After dissolution of fibrin, protein concentration was determined by optical absorption at 280 nm. The microfiltration assay displayed a high correlation with the total clottable protein method (R = 0.95), and fibrinogen antigen (r = 0.96). Correlation with clotting time assays, and PT-derived fibrinogen in 150 clinical plasma samples was in the range of r = 0.84 to r = 0.97. Intraassay and day-to-day variability of the assay was comparable to the conventional total clottable fibrinogen assay. The novel microfiltration assay appears to be well suited for measurement of large series of samples for calibration, screening purposes, and clinical trials.
Clinical Prediction of Deep Vein Thrombosis in Patients with Leg Symptoms
Authors: Joseph Lawrence , Abenhaim Lucien , Leclerc Jacques , Kahn Susan
Published: March 2018

Symptoms and clinical signs individually are inaccurate for the diagnosis of DVT. However, when assessing patients with leg symptoms, clinicians have access to additional information, such as whether or not DVT risk factors are present that could improve the accuracy of clinical judgment. The purpose of this study was to identify which clinical variables best predict DVT, and to use these variables to create a clinical prediction index for DVT. We studied 271 university hospital patients with a first episode of symptomatic, clinically suspected DVT. The prevalence of DVT was 27%, of which 71% were proximal. At baseline, information was collected on demographic features, comorbidity, and symptoms and signs. A Bayesian model selection strategy was used to estimate the logistic regression model that best predicted DVT. Male sex [OR = 2.8 (1.5, 5.1)], orthopedic surgery [OR = 5.4 (2.2, 13.6)], warmth [OR = 2.1 (1.2, 3.9)] and superficial venous dilation on exam [OR = 2.9 (1.4, 5.7)] were independent predictors of DVT. Using the model, a clinical prediction index that categorized patients into different levels of DVT risk was created, and was useful in a theoretical strategy aimed to limit the need for contrast venography in patients with suspected DVT, such that 96% of study patients could have avoided contrast venography. This index should be evaluated prospectively in other patient populations.
Platelet Contribution to Leukotriene Production in Inflammation: In Vivo Evidence in the Rabbit
Authors: Celardo Antonio , Dell’Elba Giuseppe , Manarini Stefano , Mironov Alexander , Gaetano Giovanni , Cerletti Chiara , Evangelista Virgilio
Published: March 2018

The contribution of platelets to arachidonic acid transcellular metabolism may represent an important pathway of leukotriene (LT) production. The aim of this study was to investigate the role of platelets on LT production in an acute inflammatory model in the rabbit. Preliminary experiments showed that rabbit whole blood (5 ml) stimulated in vitro with the calcium ionophore A23187 produced LTB4 (52.7 ± 13.9 ng) and the mixed 5,12-DiHETE (7.25 ± 0.75 ng). In A23187-stimulated thrombocytopenic blood, LTB4 was significantly reduced to 19.5 ± 8.6 ng and 5,12-DiHETE was undetectable. Peptido-LTs were undetectable in both conditions. In experiments using washed cells, addition of thrombin-activated platelets to fMLP-activated PMN resulted in the appearance of 5,12-DiHETE and in more than twofold increase of LTB4 synthesis. When 3H-arachidonic acid-labelled platelets were mixed with unlabelled PMN and challenged with fMLP and thrombin, radioactive LTB4 and 5,12-DiHETE were produced, indicating that platelet-derived arachidonic acid was utilized by PMN 5-lipoxygenase. Intravenous infusion of fMLP (2.5 nmol/kg/min) in the rabbit induced marked granulocytopenia, thrombocytopenia and increased TxB2 plasma concentrations within 3 min. Electron microscopy of lungs showed morphologically activated and aggregated platelets occluding the capillary lumen. Activation and recruitment of circulating cells was accompanied by the production of LTB4 (peak levels at 1 min: 30.0 ± 9.5 ng/ml) and LTE4 (peak levels at 10 minutes: 77.8 ± 11.6 ng/ml). The areas under the blood concentrationtime curve (AUC, ng min/ml) corresponded to 812 ± 182 and 3692 ± 658 for LTB4 and LTE4, respectively. In immunologically thrombocytopenic rabbits, the AUC for LTB4 (86.0 ± 23.0) and LTE4 (1165 ± 542) were both significantly different from controls while in rabbits treated with an anti-leukocyte antiserum, both LTB4 and LTE4 were similar to controls. This experimental model provides in vivo evidence that platelets, involved in an acute inflammatory event contribute to the transcellular production of LTs.
The Risk of Thrombosis in Patients with Lupus Anticoagulants Is Predicted by their Specific Coagulation Profile
Authors: Finazzi Guido , Norbis Francesca , Marziali Stefana , Marchioli Roberto , Barbui Tiziano , Galli Monica
Published: March 2018

Lupus anticoagulants belong to the family of antiphospholipid antibodies. They include two phospholipid-dependent inhibitors of coagulation that may be distinguished on the basis of specific coagulation profiles generated from the comparison of the ratios of the Kaolin Clotting Time (KCT) and the dilute Russell’s Viper Venom Time (dRVVT): when the ratio of the KCT exceeds that of the dRVVT, the plasma is allocated to the “KCT” coagulation profile, when the opposite occurs, the plasma is defined to belong to the “dRVVT” coagulation profile group. We prospectively followed-up a historical cohort of 100 consecutive patients with lupus anticoagulants referred to our Institution between January 1988 and October 1997 to investigate the relationship between their coagulation profile at diagnosis and the development of thrombosis during a median follow-up time of 37.5 months (range 1-115 months). Fifty-six patients were allocated to the “dRVVT” coagulation profile, whereas the other 44 displayed the “KCT” profile. Lupus anticoagulants were transient in 17 patients, without differences between the two groups. None of these patients developed clinical events before disappearance of the phospholipid-dependent inhibitors of coagulation. The 83 cases with persistent lupus anticoagulants consistently displayed the same coagulation profile they had been allocated to at entry. Fourteen patients developed 18 thromboembolic events during the follow-up, with an overall rate of thrombosis of 4.2% patients-year. Twelve of them belonged to the “dRVVT” coagulation profile, whereas the other 2 to the “KCT” profile (p = 0.03). The “dRVVT” coagulation profile gave an odds ratio of thrombosis of 5.25 (95% confidence interval [C.I.]: 1.17-23.50). Ten of the 14 patients who developed thrombosis during follow-up had already experienced thrombosis: a previous thrombotic event caused an odds ratio of recurrency of 2.72 (95% C.I.: 0.85-8.73) (p = 0.09). By multivariate analysis, the “dRVVT” coagulation profile was still associated with a trend to a higher risk of thrombosis, but the difference did not reach statistical significance. Increased levels of anticardiolipin antibodies (> 40 GPL and/or MPL units) were found in all the 14 patients (p = 0.0064). The “KCT” coagulation profile was significantly associated (p = 0.005) with moderate thrombocytopenia (platelets 50-150 × 109/l). Neither profile was found to represent a risk factor for the development of recurrent miscarriages, neoplastic diseases and death. In conclusion, the “dRVVT” profile appears to have predictive value with respect to the thrombotic complications suffered by patients with antiphospholipid antibodies.
Blast Cell-surface and Plasma Soluble Urokinase Receptor in Acute Leukemia Patients: Relationship to Classification and Response to Therapy
Authors: Alitalo Riitta , Stephens Ross , Vaheri Antti , Mustjoki Satu
Published: March 2018

Plasminogen activation in leukemia has been less well characterized than in other malignancies. However, the increased tendency to bleeding and tissue infiltration by leukemic cells are processes in which plasminogen activation may be involved. We have examined plasma and the peripheral blood mononuclear cell fraction from 80 patients including 53 patients with newly diagnosed acute leukemia and 27 patients with other hematological disorders as well as 21 healthy controls. In 28 of 29 examined patients with acute myeloid leukemia (AML) and in two of three patients with hybrid leukemia we found urokinase receptor (uPAR) on the cell surface, while most (7/9) samples from patients with acute lymphoblastic leukemia (ALL) were negative for uPAR. The plasma mean value for soluble uPAR (suPAR) was significantly elevated in patients with AML and ALL. In AML the highest values were found in patients who had residual disease after several cycles of chemotherapy. Compared to controls the uPA antigen levels in patient plasmas were elevated and decreased along with uPAR during treatment. Our results suggest that cell surface uPAR may be a useful marker for leukemia classification and in our material a high level of plasma suPAR correlated with resistance to chemotherapy in AML.
Clotting Activation after Transjugular Intrahepatic Portosystemic Stent Shunt
Authors: Basili Stefania , Manuela Merli , Ferro Domenico , Lionetti Raffaella , Rossi Elisabetta , Riggio Oliviero , Valeriano Valentina , Capocaccia Livio , Violi Francesco
Published: March 2018

Background and Aim. Aim of the study was to investigate the behaviour of clotting system in peripheral circulation of cirrhotic patients undergoing transjugular intrahepatic portosystemic stent shunt (TIPS). Methods. Clotting variables and endotoxemia were measured 48 h and 30 days after TIPS in patients randomised to receive heparin or not. Results. Forty-eight hours after TIPS, a significant increase of prothrombin fragment F1+2 was observed; such increase was less evident in patients given heparin. Similar findings were observed for endotoxemia, which, however, was not affected by heparin treatment. Thirty days after TIPS procedure prothrombin fragment F1+2 and endotoxemia returned to baseline values independently of the treatment given. Conclusion. This study shows that TIPS is associated with an increase of clotting activation which might contribute to acute thrombosis observed after this procedure.
Prediction of Deep Vein Thrombosis after Elective Hip Replacement Surgery by Preoperative Clinical and Haemostatic Variables: The ECAT DVT Study
Authors: Haverkate Frits , Thompson Simon , Turner Rebecca , Bertina Rogier , Turpie Alexander , Mannucci Pier , Lowe Gordon ,   on behalf of the ECAT DVT Study Group
Published: March 2018

The European Concerted Action on Thrombosis (ECAT) DVT Study was a collaborative study of preoperative haemostatic tests in prediction of DVT (diagnosed by routine bilateral venography) after elective hip replacement. 480 patients were recruited in 11 centres across Europe. Clinical risk factors were assessed, and stored citrated plasma aliquots were centrally assayed for 29 haemostatic factors according to the ECAT methodology. 120 (32%) of 375 evaluable patients had DVT, and 41 (11%) had proximal DVT. Among clinical variables, DVT was significantly associated with increased age, obesity, and possibly non-use of stockings. Of the 29 haemostatic factors, mean preoperative levels were significantly higher in patients with subsequent DVT (on univariate analyses) for factor VIII activity, prothrombin fragment F1+2, thrombin-antithrombin complexes, and fibrin D-dimer; and significantly lower for APTT and APC sensitivity ratio. Factor V Leiden was also associated with DVT. Most of these variables were also associated with age, while D-dimer was higher in patients with varicose veins. On multivariate analyses including clinical variables, only a shorter APTT (locally but not centrally performed) and APC resistance showed a statistically significant association with DVT.

We conclude that (a) DVT is common after elective hip replacement despite prophylaxis; (b) the study provides some evidence that DVT is associated with a preoperative hypercoaguable state; and (c) preoperative haemostatic tests do not add significantly to prediction of DVT from clinical variables, with the possible exception of APC resistance.
Further Studies on the Mechanism for the Antithrombotic Effects of Naroparcil, an Orally Active Thioxyloside Compound
Authors: Theveniau J. , Coup D. , Grégoire T. , Vaillot M. , Dupouy D. , Sié P. , Boneu B. , Millet J. , Masson P.
Published: March 2018

The antithrombotic β-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII.

The qualitative analysis of GAGs showed the presence of the ΔDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED80 values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.
Ins405AsnPro Mutation in the von Willebrand Factor Propeptide in Recessive Type 2A (IIC) von Willebrand’s Disease
Authors: Karpman D. , Isaksson C. , Kristoffersson A. C. , Lethagen S. , Schneppenheim R. , Holmberg L.
Published: March 2018

The molecular defects of the von Willebrand factor (vWF) have been studied in the patient in whom the von Willebrand disease phenotype IIC was originally described. A six nucleotide insert, AATCCC, was found in exon 11 of the vWF gene, predicting the insertion of the amino acids asparagine and proline between phenylala-nine 404 and threonine 405 of the vWF propeptide. The mutation was present in one allele. Analysis of amplification products derived from platelet vWF mRNA showed the other allele to be silent. The patient is thus a compound heterozygote for a null allele and the IIC allele, in accord with the recessive mode of inheritance of the IIC phenotype. Family studies indicated the IIC mutation to have occurred de novo, possibly as a result of a duplication event. In vitro mutagenesis and expression in COS-7 cells confirmed the detrimental effect of the mutation on vWF multimer assembly. Taken together with those of earlier studies the present findings suggest that the IIC phenotype may well be exclusively caused by mutations which result in changes of the amino acid sequence in certain regions of the vWF propeptide. Although in the recently revised classification of von Willebrand’s disease variants, the IIC type is included in the 2A category, obviously it constitutes a very distinct subtype.
Weight-Loss Induced Changes in Plasma Factor VII Coagulant Activity and Relation to the Factor VII Arg/Gln353 Polymorphism in Moderately Obese Adults
Authors: Folsom Aaron , Shahar Eyal , Tsai Michael , Jeffery Robert , Wing Rena , Pankow James
Published: March 2018

Elevated plasma factor VII coagulant activity (factor VIIc) may be an independent risk factor for coronary heart disease. Several cross-sectional studies suggest that a polymorphism of the factor VII gene (ArgGln353) interacts with plasma triglyceride level in determining factor VIIc, but prospective data are lacking. Factor VII genotype, factor VIIc, and triglyceride level were measured in moderately obese adults aged 25 to 45 who underwent a six-month clinical trial to evaluate strategies for weight loss. A total of 48 men and 50 women who experienced substantial weight loss (mean: 10 kg) provided samples for genetic analysis. Overall, 78% of participants were homozygous for the Arg353 allele, while the remaining 22% were heterozygous (Arg/Gln353). At the baseline examination, heterozygotes had lower mean factor VIIc than Arg353 homozygotes (92% vs. 112%; p<0.001), and genotype explained 18% of the variance of factor VIIc. Average six-month weight loss was similar in both genotypes; mean reductions in factor VIIc following weight loss were greatest among Arg353 homozygotes with high initial values (>120%). Cross-sectional and longitudinal associations between plasma factor VIIc and triglyceride level were not dependent on genotype. These data confirm that the Gln353 allele is associated with lower factor VII coagulant activity in moderately obese adults, but they do not support the hypothesis that the Arg-Gln353 polymorphism interacts with plasma triglyceride level in determining factor VIIc.
Monoclonal Antibody-based Immunoassays for the Specific Quantitation of Rat PAI-1 Antigen and Activity in Biological Samples
Authors: Ngo Thu-Hoa , Verheyen S. , Knockaert I. , Declerck P. J.
Published: March 2018

Two enzyme-linked immunosorbent assays (ELISAs) for the quantitation of rat plasminogen activator inhibitor-1 (PAI-1) antigen and activity, respectively, in biological fluids were developed using monoclonal antibodies raised against recombinant rat PAI-1. These assays had a lower limit of sensitivity in plasma of 0.3 and 0.15 ng/ml, respectively. The intra-assay, inter-assay and inter-dilution coefficients of variation were 9, 14 and 9%, respectively, for the antigen assay and 8, 17 and 13%, respectively for the activity assay. Assay recoveries of recombinant rat PAI-1 (5 to 20 ng/ml) added to plasma were 73 to 88% and 89 to 93% for the antigen and the activity assay, respectively. The level of PAI-1 antigen in rat plasma was 1.8 ± 0.9 ng/ml (mean ± SD, n = 18), with a corresponding value of 1.0 ± 0.5 ng/ml for PAI-1 activity. In lysed platelet-rich rat plasma PAI-1 antigen was 6.0 ± 1.0 ng/ml (n = 8) and PAI-1 activity was 2.3 ± 0.4 ng/ml (n = 8). Endotoxin injection (0.5 mg/kg) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rat plasma, eventually resulting in a 100- to 200-fold increase (p <0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.63, n = 18, p <0.01) and in plasma from endotoxin-treated rats (r = 0.90, n = 35, p <0.001). Application of these assays for the analysis of gel filtration experiments of plasma from endotoxin-treated rats demonstrated that PAI-1 antigen eluted as two peaks (with corresponding Mr of ~430 kDa and 61 kDa) whereas PAI-1 activity eluted as a single peak corresponding with the high molecular weight antigen form.

Thus, these unique assays allowing the specific determination of rat PAI-1 antigen and rat PAI-1 activity may constitute important tools for further investigations on the pathophysiological role of PAI-1 in a variety of experimental rat models.
Multiple Inhibition of Platelet Activation by Aurintricarboxylic Acid Prevents Vascular Stenosis after Endothelial Injury in Hamster Carotid Artery
Authors: Kozawa Osamu , Niwa Masayuki , Tanabe Kumiko , Ichimaru Katsuhiro , Takiguchi Yoshiharu , Yokota Masayuki , Hayashi Hideharu , Uematsu Toshihiko , Matsuno Hiroyuki
Published: March 2018

Activated platelets are instrumental in restenosis due to their role in thrombus formation. Aurintricarboxylic acid (ATA) has been reported to prevent platelet activation by inhibiting von Willebrand factor binding to platelet glycoprotein (GP) Ib. We investigated the effects of ATA in vitro and in vivo in hamsters. ATA inhibited the in vitro platelet aggregation induced by ADP, botrocetin and thrombin, but not by collagen. The IC50 values during the ex vivo platelet aggregation by ADP, botrocetin and thrombin were 8.2 ± 1.8 μM, 0.9 ± 0.4 μg/ml and 2.4 ± 0.8 unit/ml, respectively. The platelet retention time to collagen-coated beads of hamster blood samples was inhibited by ATA (0.1, 0.3 and 1.0 mg/kg per hour) in a dose-dependent manner. Continuous administration of ATA (0, 0.1, 0.3, 1.0, 3.0 and 10.0 mg/kg per h) via an infusion pump produced dose-dependent antithrombotic effects: the time to occlude the carotid artery after vascular injury with a modified catheter was prolonged. Only when infused at doses of 3.0 and 10.0 mg/kg per hour, bleeding times were significantly prolonged. The continuous treatment with ATA (1.0 mg/kg per h) using a 2ML1 Alzet infusion pump for 2 weeks, resulted in a decrease in neointimal area by 22.2 ± 6.8% when measured 2 weeks after injury induction. DNA synthesis using DDT1MF2 hamster SMCs was decreased by ATA in a dose-dependent manner. ATA reduced the number of platelets adhering on the injured area, as detected by electron microscopy. These results indicated that treatment with ATA inhibited platelet adhesion but also SMC proliferation. These observations may explain the effect of ATA on neointima formation.
Very Low Activated Factor VII and Reduced Factor VII Antigen in Familial Abetalipoproteinaemia
Authors: Mitropoulos K. A. , Nanjee M. N. , Howarth D. J. , Martin J. C. , Esnouf M. P. , Reeves B. E. A. , Miller N. E. , Cooper J. A. , Miller G. J.
Published: March 2018

Abetalipoproteinaemia is a rare disorder of apolipoprotein B metabolism associated with extremely low plasma concentrations of triglyce-ride. To discover whether the general positive association between factor VII and triglyceride levels extends to this condition, 5 patients were compared with 18 controls. All patients had a triglyceride below 100 μmol/l. Plasma unesterified fatty acid concentration was normal. Although factor IX activity was only slightly reduced (mean 88% standard) and factor IX antigen was normal, mean activated factor VII in patients was strikingly reduced to 34% of that in controls, a level similar to that found in haemophilia B. The patients’ mean factor VII activity and factor VII antigen were also significantly reduced to 54% and 63% of those in controls, respectively. Mean factor XI activity and tissue factor pathway inhibitor activity were reduced in patients to 70% and 75% of control values respectively, while factor XII, factor XI antigen, factor X, prothrombin and protein C were normal.
Treatment with a GPIIb/IIIa Antagonist Inhibits Thrombin Generation in Platelet Rich Plasma from Patients
Authors: Keularts I. M. L. W. , Béguin S. , Zwaan C. de , Hemker H. C.
Published: March 2018

Infusion of the GPIIb/IIIa-inhibitor MK383 inhibits thrombin generation in platelet rich plasma by interfering with the production of platelet procoagulant phospholipid exposure. The effect is similar to that of 0.2 U/ml of heparin. Heparin infusion, well known to inhibit thrombin generation by fostering antithrombin activity, inhibits the formation of platelet-derived procoagulant microparticles, probably by decreasing the formation of free thrombin, which, under our circumstances, is the main platelet activator.
Proteolysis of Tissue Factor Pathway Inhibitor (TFPI) by Plasmin: Effect on TFPI Activity
Authors: Li Anguo , Wun Tze-Chein
Published: March 2018

An important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI). TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway. Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plaminogen activators. In this study, the interaction of plasmin with recombinant E. coli-derived TFPI (rTFPI) was examined. Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities. Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257. Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity. These changes were abolished by addition of aprotinin, an inhibitor of plasmin. These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated. The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.
Quantitative Analysis of von Willebrand Factor and Its Propeptide in Plasma in Acquired von Willebrand Syndrome
Authors: Boertjes Ria , van Mourik Jan , van Genderen Perry
Published: March 2018

Measurement of the von Willebrand factor (vWF) propeptide, also known as von Willebrand antigen II, has been suggested to be helpful in the discrimination of congenital von Willebrand disease type I from type 2 and in assessing the extent of activation of the endothelium. We performed a quantitative analysis of mature vWF and its propeptide in plasma in 8 patients with acquired von Willebrand syndrome (AvWS) and in 20 normal individuals. Mature vWF levels were significantly lower in AvWS as compared with normal individuals (13.4 ± 3.5 vs 35.6 ± 3.3 nM, p <0.001). In contrast, propeptide levels were significantly higher in AvWS (11.4 ± 1.1 vs 4.7 ± 0.2 nM, p < 0.001), probably reflecting a compensatory increase in vWF synthesis or increased perturbation of the endothelium in AvWS. After treatment with DDAVP, propeptide and mature vWF levels rose 5-fold in AvWS, whereas propeptide levels were not altered by the infusion of a vWF concentrate or treatment with high dose intravenous immunoglobulins, indicating that plasma propeptide levels are a reliable reflection of vWF synthesis. Measurement of propeptide levels may provide additional information in AvWS as to whether decreased levels of mature vWF in the circulation are due to a decrease in synthesis or due to an accelerated removal of vWF from the circulation.
Intratracheal Administration of Recombinant Human Factor IX (BeneFix™) Achieves Therapeutic Levels in Hemophilia B Dogs
Authors: Russell K. E. , Read M. S. , Bellinger D. A. , Leitermann K. , Rup B. J. , McCarthy K. P. , Keith J. C. , Khor S. P. , Schaub R. G. , Nichols T. C.
Published: March 2018

The purpose of this paper was to establish proof of concept for administration of human recombinant F.IX (rF.IX) by inhalation for therapy of hemophilia B. The pharmacokinetics of intratracheal (IT) administration of rF.IX was studied in nine hemophilia B dogs randomized into 3 groups that received 200 IU/kg IT, 1000 IU/kg IT, or 200 IU/kg intravenously (IV). IT rF.IX produced therapeutic levels of F.IX antigen and activity and the pharmacokinetic parameters were consistent with a slow release from a depot site within the lungs. Bio-availability compared to IV administration was 11% for 200 IU/kg IT and 4.9% for 1000 IU/kg. The whole blood clotting time began to shorten at 2 h but F.IX bioactivity was not detected until 8 h post infusion in both IT groups. In all groups, F.IX activity was detected through 72 h post administration. These data demonstrate that biologically active rF.IX can reach the systemic circulation when given IT. Aerosolization of rF.IX may provide a needle-free therapeutic option for delivery of rF.IX to hemophilia B patients.
Neutrophil Proteases Can Inactivate Human PAR3 and Abolish the Co-receptor Function of PAR3 on Murine Platelets
Authors: Cumashi Albana , Ansuini Helenia , Celli Nicola , Blasi Antonio De , O’Brien Peter , Brass Lawrence , Molino Marina
Published: March 2018

Three members of the protease-activated receptor family, PAR1, PAR3 and PAR4, are activated when thrombin cleaves the receptor N-terminus, exposing a tethered ligand. Proteases other than thrombin can also cleave PAR family members and, depending upon whether this exposes or removes the tethered ligand, either activate or disable the receptor. For example, on human platelets PAR1 is disabled by cathepsin G, although aggregation still occurs because cathepsin G can activate PAR4. The present studies examine the interaction of cathepsin G and a second neutrophil protease, elastase, with PAR3 using two model systems: COS-7 cells transfected with human PAR3 and mouse platelets, which express PAR3 and PAR4, but not PAR1. In contrast to human platelets, cathepsin G did not aggregate murine platelets, and prevented their activation only at low thrombin concentrations. Elastase had no effect on thrombin responses in mouse platelets, but when added to COS cells expressing human PAR3, both cathepsin G and elastase prevented activation of phospholipase C by thrombin. Notably, this inhibition occurred without loss of the binding sites for two monoclonal antibodies that flank the tethered ligand on human PAR3. We therefore conclude that 1) exposure to cathepsin G disables signaling through human PAR3, and prevents murine PAR3 from serving its normal role, which is to facilitate PAR4 cleavage at low thrombin concentrations, 2) elastase disables human, but not murine, PAR3, 3) in contrast to human PAR4, mouse PAR4 will not support platelet aggregation in response to cathepsin G, and 4) the inactivation of human PAR3 by cathepsin G and elastase involves a mechanism other than amputation of the tethered ligand domain. These results extend the range of possible interactions between PAR family members and proteases, and provide further support for species-specific differences in the interaction of these receptors with proteases other than thrombin.
Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly
Authors: Celie Patrick , Kolkman Joost , Lenting Peter , Mertens Koen
Published: March 2018

Introduction

The activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.

Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.

During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.
Fibrinolysis and the Contact System: A Role for Factor XI in the Down-Regulation of Fibrinolysis
Authors: Meijers Joost , Bouma Bonno
Published: March 2018

Introduction

Exposure of blood to negatively-charged surfaces, such as collagen, kaolin, or glass, results in the activation of the contact system of the intrinsic pathway of coagulation. Prekallikrein, factor XII, high molecular weight kininogen, and factor XI are the proteins involved in this contact reaction. The assembly of these components on a negatively-charged surface leads to the activation of factor XI, thereby propagating the intrinsic coagulation pathway. Simultaneously, several other reactions occur, such as the activation of factor VII and the initiation of the fibrinolytic system, kinin-forming pathway, and renin-angiotensin pathway.

The first step in the contact phase is to bind factor XII to the negatively-charged surface, making it highly susceptible for proteolysis by kallikrein.1-3 Activated factor XII (α-factor XIIa) is formed in a process that may involve autoactivation.4-7 Prekallikrein is bound to high molecular weight kininogen in plasma. High molecular weight kininogen associates with a negatively-charged surface, thereby localizing prekallikrein to the surface. Limited proteolysis by α-factor XIIa converts prekallikrein to kallikrein. Kallikrein can dissociate from the surface and act on surface-bound factor XII at distant sites, thereby propagating the reciprocal cycle.7 Factor XI circulates plasma in a complex with high molcular weight kininogen. High molecular weight kininogen links factor XI to a negatively charged surface where it is activated by surface bound:α-factor XIIa. Although the in vivo, activating, negatively-charged surface is unknown, assembly and activation of the contact system on biological membranes of endothelial cells, platelets, neutrophils, and monocytes can take place, suggesting that these surfaces are the actual activating surfaces in vivo.8

The physiological significance of the contact system in blood coagulation remains unclear, however, because a deficiency of factor XII, prekallikrein, and high molecular weight kininogen does not result in a bleeding disorder. In contrast, patients deficient in factor XI, most common among Ashkenazi Jews, do suffer from variable bleeding abnormalities, especially from tissues with high local fibrinolytic activity (e.g., urinary tract, nose, oral cavity, tonsils).9,10 This suggested that there was an alternative route for the activation of factor XI, and recently, such a route was described.11,12 Thrombin was found to activate factor XI even in the absence of a negatively-charged surface,11-15 and factor XI was shown to play a role in the downregulation of fibrinolysis.16 In this article, the role of the contact system, with an emphasis on factor XI in the regulation of the fibrinolytic system, will be described.