The use of hemostatic agents for patients with platelet function related disorders causing impaired clotting is predominantly monitored via clinical parameters to date. For HPS patients, which suffer from a platelet storage pool disease, up to now mainly desmopressin and/or platelet concentrates have been administered as therapeutic agent in case of bleeding or surgery, with variable success. A more recent option is rFVIIa (NovoSeven®, N7), but there are few reports of its use in HPS patients. Before administering, it would be of therapeutical interest to know the maximum efficacy and the patient's individual response. We investigated the hemostatic efficacy of rFVIIa in a patient with HPS-1 (63 y; 2 mutations: 972delC, T322N), who was undergoing tooth extraction. rFVIIa (90 U/kg body weight bolus) was administered 30 minutes before the procedure. Citrated blood samples were taken before, 15 minutes after rFVIIa administration and after the dental treatment was completed. Platelet rich plasma was generated by centrifugation (750g, 5 min) and the samples assayed using a fluorometric thrombin generation test (TGT), thrombelastography (RoTEG) and flow cytometry (FACS). The TGT was started adding Ca 16,7 mM and fluorogenic substrate. RoTEG was used with Ca, N7, tissue factor separately and combined. From the pre-substitution samples a reference value with rFVIIa, 80 U/ml added in vitro, and a dose-response curve from 0 to 400 U/ml were measured. N7 recovery was evaluated from pre-, post-substitution and the reference values. The TGT showed a presubstitutional residual activity of 50 % of the maximum ETP value, and 100% after substitution. TGT response to N7 was dose-dependent and entered a plateau at 90 % of the value after substitution, from 80 U/ml N7 on. The effect of subsituted rFVIIa persisted 2h after the extraction. Other TGT parameters (peak, time to peak, lag time) changed concordantly. RoTEG changes were most pronounced for Ca 12 mM alone as a trigger, which showed a transient reduction of clotting time and clot formation time after N7 substitution. FACS showed a 2.2fold CD62P increase indicating platelet activation after administration. Altogether, the TGT parameter changes corresponded best to the clinical outcome, as no bleeding events were observed during and 2h after the dental procedure. Thrombin generation may thus be useful to evaluate and predict N7 therapeutic efficacy in HPS patients. More HPS patients have to be treated to confirm these findings.

Summary.  Background: Limited data exist on the clinical manifestations of homozygous factor (F)V:G1691A mutation (FV Leiden) and the impact of environmental and genetic risk factors. Objectives: To assess the contribution of these factors on the thrombophilic phenotype. Patients and methods: In a retrospective multicenter cohort study 165 individuals with homozygous FV:G1691A mutation, of whom 129 had previous venous thromboembolism (VTE), were included. To study the role of environmental risk factors, patients were compared by the use of a standardized questionnaire to 165 sex‐ and age‐matched individuals (reference group A); of these, two had previous VTE. To assess the role of genetic risk factors, factor (F)II:G20210A and MTHFR:C677T were determined in individuals homozygous for FV:G1691A and in 177 healthy individuals without previous VTE (reference group B). Results: The first VTE occurred significantly earlier in women (median age 25 years) than men (35.5 years). In 81% of women and 29% of men an environmental risk factor was present before first VTE. Oral contraceptives increased the risk of thrombosis 4‐fold [odds ratio (OR) 4.0, 95% confidence interval (CI) 1.7, 10.4] in women with homozygous FV:G1691A. Postoperative and post‐traumatic VTE as first manifestation occurred in 13% and 15% of surgical/traumatic events in patients and in 0.7% and 1.8% in reference group A, respectively (OR 19.7, 95% CI 2.5, 154 and OR 9.2, 95% CI 1.1, 79.4). Heterozygous FII:G20210A was more prevalent in symptomatic patients (11.7%) compared with reference group B (2.8%, OR 4.6, 95% CI 1.6, 13.2). The prevalence of homozygous MTHFR:C677T genotype was similar in patients and reference group B. Conclusions: Our study supports the concept of thrombophilia as a multifactorial disorder. The knowledge of coexisting factors predisposing to VTE is useful for medical advice for primary and secondary prophylaxis in these patients.

ACE displays potent vasoconstrictive effects, attenuation of fibrinolysis, and platelet activation and aggregation, thus possibly promoting venous thromboembolism (VTE). The ACE gene contains an insertion (I) or deletion (D) polymorphism accounting for 50% of the variation in serum ACE concentration. To evaluate the role of the I/D polymorphism in VTE, its prevalence was determined in 931 patients with VTE and 432 blood donors. The prevalence of the DD genotype was 27.6% in patients and 21.3% in controls (OR 1.4; p < 0.02). In multivariate analysis there was a trend of the DD genotype to be an independent risk factor (OR 1.4; p = 0.08). No differences in DD genotype prevalence according to exogenous risk factors were found. Coinheritance of FV G1691A, PT G20210A mutation, and PS deficiency with the DD genotype increased the relative risk of VTE. Thus, the ACE DD genotype is a moderate risk factor of hereditary thrombophilia. Exogenous risk factors did not alter the manifestation of VTE among carriers of the DD genotype, whereas coinheritance of the DD genotype with the aforementioned defects increased the risk for VTE considerably.

One of the most frequent causes of morbidity and mortality among Caucasians is the development of thromboembolic disorders. Besides circumstantial influences, patients carrying prothrombotic gene defects are at risk of developing VTE. Among others, the protein C anticoagulant pathway is an important mechanism regulating thrombin generation and the blood clotting response. Therefore deficiencies of members of the protein C anticoagulant pathway may cause severe thrombotic complications. Both protein C and activated protein C (APC) bind to human endothelial cell protein C receptor (EPCR), a newly described member of the protein C anticoagulant pathway [1, 2]. EPCR is located on the surface of endothelial cells of larger vessels, especially of arteries [3, 4], and one of its physiological functions seems to be the enhancement of protein C activation by the thrombin-thrombomodulin complex in an EPCR concentration dependent fashion [5]. On the other hand, EPCR seems to inhibit the APC anticoagulant activity [6], which in turn could cause increased thrombotic tendency 17, 8]. Recently, a 23 bp insertion in exon 3 of the EPCR gene at position 7116 generating a duplication of the 23 bases preceding the insertion point has been described [9]. The mutation generates a STOP codon at the sixth codon downstream of the insertion point. The predicted receptor is truncated missing a part of exon 3 and the whole exon 4. This prompted us to evaluate the frequency of EPCR insertion in patients with VTE.

In order to assess inhibitor development in previously untreated patients (PUPs) with severe (factor VIII [FVIII]<1%) and moderate (FVIII 1 to 5%) hemophilia A, a prospective study was initiated in 1976. During the 23-year study period, 72 hemophiliacs were frequently exposed prophylactically or on demand to plasma-derived (pd) (n = 51) or recombinant FVIII (rFVIII) (n = 21) concentrates (median 270 exposure days [ED]). Inhibitor testing was performed before the first exposure and at regular intervals thereafter. Of the 72 hemophilia A patients, 22 (32%) developed an inhibitor after 15 ED in median (range 4 to 195); 17 (77%) were high responders (>5 Bethesda Units [BU]), and the remaining 5 patients (23%) were low responders (>0.6 to 5 BU). The severely affected patients (n = 46) showed a significantly higher frequency of inhibitor formation (43%) than did the moderate ones (8%). Comparing the severely affected patients receiving pd products exclusively (n = 35) with those treated with recombinant concentrate (n = 11), 37% of the pd group developed a high-titer inhibitor (>5 BU, median 290 ED in noninhibitor patients) and 36% of the recombinant group (median 49 ED in the noninhibitor patients). However, the exposure status of the recombinant noninhibitor patients is rather low and therefore remains a high risk of developing further inhibitors in the future. The mutation type profile revealed no difference between the pd- and the recombinant-treated patients.

Within the last decade, various genetic defects of proteins regulating blood coagulation, particularly those affecting the physiological anticoagulant systems, have been well established as risk factors of cerebrovascular disease in adults [8;22;23]. Besides the high thrombotic risk reported in patients with homozygous FV:R506Q mutation, homozygous protein C deficiency, homozygous protein S deficiency and homozygous homocysteinuria due to cystathionine-ß-synthase (CBS) deficiency, a high risk of early thrombotic onset is observed in patients with heterozygous anti-thrombin deficiency, heterozygous protein C deficiency of the so-called dominant type, and heterozygous protein S deficiency [18;29;40].

After a first episode of spontaneous venous thromboembolism (VTE), the risk of recurrence persists for many years. However, comprehensive data about the risk of recurrence in pediatric patients have hitherto not been reported. Thus, this study evaluated the risk of recurrent VTE among children in relation to the presence of single or combined-inherited and/or acquired causes of thrombophilia. A total of 301 patients aged neonate to 18 years (median, 6 years) who were referred for an objectively confirmed first episode of spontaneous VTE were followed prospectively for a median time of 7 years (range, 6 months to 15 years) after withdrawal of anticoagulation. All patients were studied for established acquired and inherited causes of thromboembolism. With reference to all 301 patients, one single prothrombotic risk factor was found in 176 subjects (58.5%), whereas combined defects were found in 20.6% (n = 62). Recurrent VTE occurred in 64 patients (21.3%) within a median time of 3.5 years (range, 7 weeks to 15 years) after withdrawal of anticoagulation, with a significantly shorter cumulative thrombosis-free survival in children carrying combined defects (P <.0001; chi-square, 42.2). The factor V G1691A mutation was present in the majority of patients with recurrent VTE. Including genetic defects, gender, and acquired risk factors, multivariate analysis showed that only the presence of prothrombotic defects increases the risk of recurrent VTE (single defect: odds ratio [OR], 4.6; 95% confidence interval [CI], 2.3-9.0; P <.0001; combined defect: OR, 24.0; 95% CI: 5.3-108.7; P <.0001). As a consequence of the data presented here, it is suggested that screening for genetic risk factors be done among pediatric patients with VTE.

Article The Role of Factor XIII Val34Leu Polymorphism in Thrombofic Disease. Die Rolle des Faktor XIII Val34Leu Polymorphismus bei thrombotischen Erkrankungen was published on January 1, 2001 in the journal Journal of Laboratory Medicine (volume 25, issue 7-8).

Venous thrombosis is a multifactorial disorder with an incidence of about 1–1.6 per 1000 population. In addition to the well-known risk factors for thrombosis (lack of AT or protein S/C, APC resistance or prothrombin mutation), other congenital and acquired disorders are also associated with an increased tendency to thromboembolic complications.

Zusammenfassung: Venöse thromboembolische Erkrankungen ereignen sich bei ca.l von 1000 Individuen jährlich.Meist handelt es sich dabei um ein multifaktorielles Geschehen, das durch Zusammenwirken erworbener bzw.exogener Risikofaktoren einerseits sowie genetisch bedingter Veränderungen andererseits verursacht ist.In den letzten Jahren wurden mehrere Risikofaktoren der hereditären Thrombophilie identifiziert, die inzwischen als etabliert gelten.Daneben gibt es jedoch eine Reihe weiterer genetischer Defekte, deren Beteiligung bei der Entstehung venöser Thrombosen wahrscheinlich oder zumindest theoretisch denkbar ist.In diesem Überblick werden als solche Lipoprotein (a), Thrombomodulin, Fibrinogen, der Thrombin-aktivierbare Fibrinolyse Inhibitor (TAFI), Gewebefaktor (Tissue Factor) sowie der Endothelzell-Protein C Rezeptor (EPCR) dargestellt, ihre biochemischen Eigenschaften sowie physiologischen Funktionen zusammengefaßt und bekannte Mutationen

Summary Background: The endothelial cell protein C receptor (EPCR) enhances protein C activation by the thrombin-thrombomodulin complex. As evidence is accumulating that EPCR is an important component of the protein C anticoagulant pathway, polymorphisms in the EPCR gene might be candidate risk factors predisposing to venous thromboembolism (VTE). Recently, a 23bp insertion in exon 3 of the EPCR gene has been identified, which duplicates the preceding 23 bases and results in a STOP codon downstream from the insertion point. However, the clinical significance of this mutation in VTE remains to be clarified. Methods and Results: In this study we evaluated the EPCR 23bp insertion in 889 patients with documented VTE and in 500 healthy controls. The prevalence of the EPCR insertion among patients was 0.1%, which was not significantly different compared to controls (0.6%, p = 0.1). Conclusions: Our findings showed that the EPCR 23bp insertion is very rare in both patients with VTE and the general population and failed to support an association between the EPCR 23bp insertion and an increased risk of VTE.

Elevation of serum lipoprotein (a) (Lp[a]) is a known risk factor predisposing to cardiovascular and cerebrovascular disease. However, little is known about the role of increased Lp(a) in venous thromboembolism (VTE). This study evaluated the role of Lp(a) among a panel of established hereditary thrombogenic defects in patients with VTE. A total of 685 consecutive patients with at least one episode of VTE and 266 sex- and age-matched healthy controls were screened with regard to activated protein C resistance, protein C, protein S, and antithrombin deficiency, elevated serum levels of Lp(a), and the factor V G1691A, MTHFR C677T, and prothrombin G20210A mutations. Elevated Lp(a) levels above 30 mg/dL were found in 20% of all patients, as compared to 7% among healthy controls (P < .001, odds ratio [OR] 3.2, 95% confidence interval [CI], 1.9-5.3). The coexistence of FV G1691A and elevated Lp(a) was significantly more prevalent among patients with VTE than in the control group (7% versus 0.8%; P < .001, OR 9.8, 95% CI, 2.4-40.7). No other established prothrombotic risk factor was found to be significantly combined with increased Lp(a). These data suggest that Lp(a) concentrations greater than 30 mg/dL are a frequent and independent risk factor for VTE. Furthermore, elevated Lp(a) levels might contribute to the penetrance of thromboembolic disease in subjects being affected by other prothrombotic defects, such as FV G1691A mutation.

Eine Arteriosklerose findet sich sehr selten bei Patienten mit ausgeprägten Blutgerinnungsstörungen. Vermutlich verhindert die beeinträchtigte Interaktion zwischen Thrombozyten, v.W.Faktor und Gefäßwandendothel bei Patienten mit vWS die Bildung und das Wachstum von Arteriosklerose.

INTRODUCTION:To evaluate the pregnancy-associated risk of venous thromboembolism and the risk of stillbirth and miscarriage a multicenter, retrospective and controlled study was conducted in women carrying the homozygous factor V Leiden mutation and in an agematched control group of women from the normal population.PATIENTS AND METHODS:In 64 homozygous (median age 44 years, range 21-75 years) and in 52 control women from five different centers data on venous thromboembolism and pregnancy outcome were obtained.RESULTS:The 64 homozygous women had in total 212 pregnancies, the 52 control women had 118 pregnancies. In homozygous women 65% of pregnancies ended with delivery of a viable infant, 15% with fetal loss (3.3% stillbirth, 12% miscarriage) and 20% by pregnancy termination. In the control women 75% of pregnancies ended with delivery of a viable infant, 12% with fetal loss (1.7% stillbirth, 10% miscarriage) and 13% by pregnancy termination. The differences were statistically not significant. Venous thromboembolism occurred significantly more often in the homozygous women, in 4.2% (9/212) during pregnancy and in 4.7% (10/212) after delivery or pregnancy termination. None of the control women had a thromboembolic episode.CONCLUSION:Our data indicate that women with homozygous factor V Leiden have a high probability for a favorable pregnancy outcome. The increased risk for venous thromboembolism during pregnancy and after delivery would favor heparin prophylaxis during and after pregnancy in women homozygous for factor V Leiden.