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36th Hemophilia Symposium: Hamburg 2005

Inge Scharrer ; Wolfgang Schramm (eds.)

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Medicine & Public Health;

Institución detectada Año de publicación Navegá Descargá Solicitá
No detectada 2007 SpringerLink


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Editor responsable

Springer Nature

País de edición

Reino Unido

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Tabla de contenidos

Factor VIII as Positive Regulator of Activated Platelets

A. Sturm; A. Obergfell; U. Walter; R. Grossmann

Deficiency of factor VIII (FVIII) as an important cofactor in the tenase coagulation factor complex causes severe bleeding,whereas high FVIII levels seem to be associated with venous and arterial thromboembolism. Resting platelets do not bind FVIII, but activated platelets bind inactivated FVIII if vWF is not present. In this study the influence of platelet bound FVIII on platelet function itself was investigated as a direct effect of FVIII on platelets has not been shown so far.Methods: The influence of platelet bound FVIII on platelet function was investigated by flow cytometric analysis of P-selectin expression (CD62P) and PAC-1 binding before and after submaximal stimulation with TRAP-6 (5 μM final concentration) and by confocal microscopy. Blood was incubated with human recombinant FVIII concentrate (Helixate, ZLB Behring,Marburg, Germany) for 5 minutes at 37°C. Results: There were no significant changes in P-selectin expression or PAC-1 binding respectively in FVIII-pretreated platelets. Stimulation of platelets with TRAP-6 increased the expression of Pselectin (445 %) and PAC-1 binding (934 %) as expected. These effects were significantly further increased when platelets were stimulated with TRAP-6 and FVIII (Pselectin 499 %, PAC-1 1626 %; Student’s t-test, p < 0.05.Values were expressed in %, related to unstimulated,Helixate buffer treated platelets). The assessment of platelet spreading by the means of confocal microscopy showed an increased spreading on fibrinogen matrix after stimulation with TRAP-6 and FVIII. This effect was higher, compared to platelets stimulated with TRAP-6 alone. Conclusions: TRAP-co-stimulated platelets seem to be positively regulation by additional FVIII. Thus a FVIIIinduced increase in platelet activation might contribute to venous and even more arterial thrombus formation in patients with high FVIII levels.

Palabras clave: Factor VIII; Platelet Function; Mean Fluorescence Intensity; Washed Platelet; Arterial Thromboembolism.

Pp. 257-262

Characterization of Three Novel Mutations in the Sodium Binding Site of Coagulation Factor X

R.F. Strey; K. Wulff; W. Schröder; F. H. Herrmann

In this study we characterized the novel mutations FX_G325R, FX_E329G and FX_G410R by biochemical methods as well as by protein modeling. For the biochemical analysis the proteins have been expressed in HEK 293 cells. The antigen activities (determined using sandwich ELISA) of the mutations FX_G325R and FX_E329G have been reduced compared to the wild type,whereas FX_G410R did not lead to reduced expression. The amidolytic and prothrombinase activities (determined using fluorogenic substrates) of all three mutants have been drastically reduced compared to the wild type. This is in accordance with reduced factor X activities in the (heterozygous) patients. The protein modeling suggests an impaired function of the sodium loop as a result of the mutations FX_G325R, FX_E329G or FX_G410R. Although only Gly410 is located in the sodium loop of factor X, all three mutations lead to a shift of the electrostatic potential in the sodium loop from the acid to the basic range. This could lead to reduced sodium or substrate binding and therefore to an impaired catalytic function of the mutants. Additionally to their influence on the electrostatic potential the mutations FX_G325R and FX_G410R may lead to steric conflicts as a result of the replacement of the small glycine by the bulky arginine. In the case of FX_G325R the side chain of Arg325 inhibits the salt bridge between Ile195 and Asp378, which is important for the formation of the catalytic active conformation of factor Xa. In the mutant FX_R410 the side chain of Arg410 may fill the binding site of the sodium ion or of the P1 residue of substrates.

Palabras clave: Electrostatic Potential; Salt Bridge; Bleeding Symptom; Heterozygous Patient; Amidolytic Activity.

Pp. 263-271

Characterization of a Mutation in the 5′ Flanking Region and a Novel IVS7 Splice site Mutation in a Patient with Severe FVII Deficiency

W. Schröder; K. Wulff; R. Tech; R. Grempler; A. Ruiz-Saez; F. H. Herrmann

The molecular basis of a factor VII deficiency was investigated in a Venezuelan patient. The five-year-old boy with a residual FVII:C of 1.7% and FVII:Ag < 8% suffered from epistaxis and easy bruising since birth. By sequence analysis a mutation <60T>C in the 5’ flanking region of the FVII gene and the novel mutation at the IVS7 donor splice site IVS7+1G>A were detected. The mutated splice site is located within the first repeat of a minisatellite region. Each repeat in this region contains a normally silent pseudo splice site. By an electrophoretic mobility shift assay (EMSA) the decrease of the binding of the nuclear factor HNF-4 by the promoter mutation -60T>C could be shown. To characterize the consequence of the mutated splice site IVS7+1G>A we constructed wildtype and mutated minigens spanning exons 7 to 8. HEK 293 cells were transfected with these constructs. In cells transfected with the mutant transcript no normal splicing occurred. Three different cryptic splice sites were used in vitro, all predicting a premature termination.

Palabras clave: Splice Site; Electrophoretic Mobility Shift Assay; Mutate Splice Site; Polymerase Chain Reac; Cryptic Splice Site.

Pp. 272-278

On the Molecular Basis of Warfarin Resistance in Rats

M. Hünerberg; S. Rost; A. Fregin; H. J. Pelz; C. R. Müller; J. Oldenburg

Palabras clave: Epoxide Reductase; Coagulation Factor Deficiency; VKORC1 Gene; Warfarin Resistance; Y139S Mutation.

Pp. 279-283

Influence of Factor V_HR2 on Endogenous Thrombin Potential and Clinical Phenotype in Factor VII Deficiency

R.F. Strey; A. Siegemund; T. Siegemund; C. Schubert; G. Schuster; K. Wulff; F. H. Herrmann

We investigated the influence of the factor V haplotype R2 (FV_HR2), defined by the mutation FV_H1299R, on the thrombin generation in plasma as well as on the clinical expression of bleeding symptoms in patients homozygous for FV_IIA294V. Due to its impaired interaction with activated protein C (APC) FV_HR2 increases the thrombin generation. Measurements of the endogenous thrombin potential in presence of high APC concentrations showed significant increased ETP values in plasma containing FV_HR2 compared to references with factor V wild type. In our measurements this effect is comparable to the increase of thrombin generation caused by FV_Leiden. This suggests FV_HR2 to be a risk factor for thrombosis similar to factor V_Leiden. On the basis of these findings, we investigated the influence of FV_HR2 on the clinical expression of bleeding symptoms in factor VII deficient patients. A moderating effect of FV_Leiden to bleeding symptoms in hemophilia A and B as well as in case of FII_Lazio has already been reported by other authors. This effect is explained by the increased APC resistance of FV_Leiden. Based on the APC resistance we expected a similar effect for FV_HR2. This was confirmed for four patients homozygous for the mutation FVII_A294V. Two of these patients did not carry FV_HR2 and show bleeding symptoms. The two asymptomatic patients are heterozygous or homozygous for FV_HR2 respectively. This indicates a moderating effect of FV_HR2 to bleeding symptoms in patients homozygous for the mutation FVII_A294V.

Palabras clave: Factor Versus; Thrombin Generation; Bleeding Symptom; Factor Versus; Endogenous Thrombin Potential.

Pp. 284-290

Splice Site Mutations Effect on the F8 mRNA Splicing

O. El-Maarri; C. Klein; J. Junen; J. Schröder; C. R. Müller; J. Oldenburg

This highlights the usefulness of the mRNA analysis for routine diagnosis of such ambiguous cases including the presence of potential cryptic splice sites and clearly demonstrates the causality of the mutations which is also important for the genetic counseling of those families.

Pp. 291-294

Species-Specific Variation of VKORC1-Activity and Resistance to Warfarin

A. Fregin; S. Rost; C. R. Müller; J. Oldenburg

Palabras clave: Rodent Species; Coumarin Derivative; Microsomal Preparation; Spiny Mouse; VKORC1 Gene.

Pp. 295-297

Various Missense Mutations in the Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) Cause Hereditary Coumarin Resistance

C. Geisen; S. Rost; G. Spohn; A. Fregin; M. Watzka; D. M. Dimichele; H. Haubelt; M. Heistinger; J. Kadar; B. Kemkes-Matthes; P. Lages; E. Lindhoff-Last; B. Luxembourg; H. Pollmann; R. Zimmermann; C. R. Müller; E. Seifried; J. Oldenburg

Mutations in VKORC1 cause coumarin resistance and are also responsible for differing dosing requirements close to the upper therapeutic range. Here we present the data in 16 patients from 14 families revealing 11 different missense mutations, six of which have not been reported previously. Our data may contribute in identifying further functional epitopes of VKORC1 and may provide a basis for a rational design of novel anticoagulants targeting VKOR in the future.

Palabras clave: Missense Mutation; Coagulation Factor; Coumarin Derivate; Functional Epitope; Epoxide Reductase.

Pp. 298-301

Establishment of an International Registry of Patients with Congenital FXIII Deficiency

V. Ivaskevicius; R. Seitz; H. P. Kohler; L. Muszbek; R. A. S. Ariens; E. Seifried; J. Oldenburg

Palabras clave: Spontaneous Abortion; Factor Xiii; Human Immune Deficiency Virus Infection; Factor Xiii Deficiency; Ristocetin Cofactor.

Pp. 302-304

The Impact of Freezing of Plasma Samples, AB0 Blood Group and Acute-Phase Reaction on Detecting Mild Factor VIII Deficiency and Increased Factor VIII Levels as a Risk Factor for Venous Thromboembolism

J. Bach; H. Haubelt; U. Seyfert; A. Vogt; P. Hoerdt; P. Hellstern

Despite identical calibration procedures, FVIII:C values are significantly greater than FVIII:AM and FVIII:Ag. This may be due to the fact that FVIII:C but not FVIII:AM and FVIII:Ag overestimates activated FVIII. The lower limits of reference ranges for FVIII entities derived from frozen and thawed plasma samples are markedly greater than reported in previous studies: FVIII:C, 73 IU/dl; FVIII:AM, 68 IU/dl; FVIII:Ag, 67 IU/dl Freezing and thawing of plasma samples reduces FVIII:C and FVIII:AM by 27% and 12%, respectively. Freezing and thawing does not influence FVIII:Ag. Fibrinogen but not FVIII plasma levels are markedly influenced by acute phase reaction as determined by hsCRP. Blood group non-0 or non-A2 individuals have substantially greater FVIII plasma levels than subjects with blood groups 0 or A2. Diagnosis of mild FVIII deficiency or increased FVIII levels as a risk factor for venous thromboembolism requires carefully established references ranges and standardized pre-analytical conditions with respect to AB0 blood group and freezing/ thawing of plasma samples. Before measuring FVIII, plasma samples should be frozen and thawed to obtain reliable results.

Palabras clave: Factor Viii; Blood Group; Acute Phase Reaction; hsCRP Level; Factor Viii Deficiency.

Pp. 305-309