Inge Scharrer

von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.

BACKGROUND Thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome are severe microvascular disorders of platelet clumping with similar signs and symptoms. Unusually large multimers of von Willebrand factor, capable of agglutinating circulating platelets under high shear stress, occur in the two conditions. We investigated the prevalence of von Willebrand factor-cleaving protease deficiency in patients with familial and nonfamilial forms of these disorders. METHODS Plasma samples were obtained from 53 patients with thrombotic thrombocytopenic purpura or hemolytic-uremic syndrome. Von Willebrand factor-cleaving protease was assayed in diluted plasma samples with purified normal von Willebrand factor as the substrate. The extent of the degradation of von Willebrand factor was assessed by electrophoresis in sodium dodecyl sulfate-agarose gels and immunoblotting. To determine whether an inhibitor of von Willebrand factor-cleaving protease was present, we measured the protease activity in normal plasma after incubation with plasma from the patients. RESULTS We examined 30 patients with thrombotic thrombocytopenic purpura and 23 patients with the hemolytic-uremic syndrome. Of 24 patients with nonfamilial thrombotic thrombocytopenic purpura, 20 had severe and 4 had moderate protease deficiency during an acute event. An inhibitor found in 20 of these patients was shown to be IgG in five of five tested plasma samples. Of 13 patients with nonfamilial hemolytic-uremic syndrome, 11 had normal levels of activity of von Willebrand factor-cleaving protease during the acute episode, whereas in 2 patients, the activity was slightly decreased. All 6 patients with familial thrombotic thrombocytopenic purpura lacked von Willebrand factor-cleaving protease activity but had no inhibitor, whereas all 10 patients with familial hemolytic-uremic syndrome had normal protease activity. In vitro proteolytic degradation of von Willebrand factor by the protease was studied in 5 patients with familial and 7 patients with nonfamilial hemolytic-uremic syndrome and was normal in all 12 patients. CONCLUSIONS Nonfamilial thrombotic thrombocytopenic purpura is due to an inhibitor of von Willebrand factor-cleaving protease, whereas the familial form seems to be caused by a constitutional deficiency of the protease. Patients with the hemolyticuremic syndrome do not have a deficiency of von Willebrand factor-cleaving protease or a defect in von Willebrand factor that leads to its resistance to protease.

Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2

Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational γ-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.

Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs

The development of factor VIII:C inhibitors remains one of the most serious complications of repeated transfusion in patients with haemophilia A. The proportion of patients affected has been reported to range from 3.6% to 25%, but these figures have been derived mainly from retrospective data and from total numbers of known haemophiliacs instead of number at true risk. The assessment here is based on a prospective study, started in 1976, on the incidence of inhibitor development in haemophiliacs born after 1970 whose FVIII or FIX activity was 5% or less, and who had received replacement therapy at least once. 46 of 63 children with haemophilia A and 13 of 17 with haemophilia B fulfilled the enrollment criteria. Inhibitors developed only in haemophilia A patients who had previously been treated with FVIII products--inhibitor concentrations were high in 12 and low in 3. Inhibitors developed in 24% (15/63) of all haemophilia A patients, and in 52% (14/27) of those with severe disease. The incidence of inhibitor development for all haemophilia patients was 39.1 per 1000 patient-years of observation. All inhibitors were first detected when patients were aged 0.08-5.2 years. The cumulative risk was 33% at age 6 years. The findings indicate that previous reports have underestimated the risk of acquiring FVIII inhibitors. Prospective, standardised studies, especially in children, are needed for the assessment of the true risk of this complication.

Human Recombinant DNA–Derived Antihemophilic Factor (Factor VIII) in the Treatment of Hemophilia A

BACKGROUND Current treatment of hemophilia A, a hereditary disorder affecting approximately 1 in 10,000 males, relies on plasma-derived factor VIII concentrates. We tested the safety and efficacy of a recombinant factor VIII preparation for the treatment of this disorder. METHODS We conducted the investigation in three stages: comparing the pharmacokinetics of plasma-derived and recombinant factor VIII, assessing the efficacy of recombinant factor VIII for home therapy, and assessing its efficacy for major surgical procedures and hemorrhage. A total of 107 subjects with hemophilia, 20 of whom had not been treated previously, enrolled in the investigation. RESULTS The in vivo recovery and elimination half-lives of recombinant factor VIII equaled or exceeded those of plasma-derived factor VIII. Seventy-six subjects participated in a home-treatment program, using recombinant factor VIII for 69 to 807 days (median, 618); home diaries of 56 subjects treated for 5 months were analyzed. Of 540 bleeding episodes, 399 (73.9 percent) required only one treatment with recombinant factor VIII. The projected annual consumption of recombinant factor VIII was similar to that of plasma-derived factor VIII concentrate. Twenty-six subjects received recombinant factor VIII for 22 surgical procedures and 10 serious hemorrhages; hemostasis was excellent in all cases. De novo formation of inhibitors occurred in only 1 of 85 previously treated subjects. Inhibitor antibodies also developed in 6 of 21 children, 20 of whom had not previously been treated; 5 had low levels (less than or equal to 7.5 Bethesda units) despite continued treatment with recombinant factor VIII. There was no evidence of new formation of antibody to foreign proteins, and recombinant factor VIII was well tolerated. CONCLUSIONS Recombinant factor VIII has biologic activity comparable to that of plasma factor VIII and is safe and efficacious for the treatment of hemophilia A.

A Positive Family History of Premature Coronary Artery Disease Is Associated With Impaired Endothelium-Dependent Coronary Blood Flow Regulation

BACKGROUND The aim of the study was to determine whether a positive family history of coronary artery disease is related to impaired coronary blood flow regulation. METHODS AND RESULTS In 150 patients with angiographically normal or minimally diseased coronary vessels, risk factors for coronary artery disease, the extent of atherosclerosis and endothelium-dependent vasomotor responses to acetylcholine, and endothelium-independent blood flow regulation by papaverine or adenosine were assessed. Coronary blood flow responses to acetylcholine were reduced in a dose-dependent manner in patients with a positive family history (P=0.030). By multivariate analysis, hypercholesterolemia (P=0.001), age (P=0.002), and a positive family history (P=0.008) remained predictors of coronary blood flow increase to acetylcholine. The extent of atherosclerotic coronary artery disease was, by multivariate analysis, an additional independent predictor of acetylcholine-induced blood flow (P=0.014), but also of endothelium-independent blood flow regulation (P=0.001). A positive family history had additive effects in addition to the other risk factors, such as hypercholesterolemia or increased age. Angiotensin-converting-enzyme genotype polymorphism had no influence either on endothelium-dependent or endothelium-independent coronary blood flow responses. However, in a subset of 28 patients, homocysteine (which is, in part, genetically determined) was inversely related to maximal acetylcholine-induced blood flow regulation (r=-0.47, P=0.012). CONCLUSIONS The results of this study demonstrate, for the first time, that a positive family history of coronary artery disease is an important predictor of impaired endothelium-dependent coronary blood flow regulation in humans. The influence of a positive family history is independent of other well known risk factors but instead aggravates endothelial vasodilator dysfunction associated with hypercholesterolemia and increased age, suggesting important interacting effects between genetic and environmental risk factors.

Multicentre evaluation of combined prothrombotic defects associated with thrombophilia in childhood

Abstract To evaluate the role of multiple established and potential causes of childhood thrombophilia, 285 children with a history of thrombosis aged neonate to 18 years (first thrombotic onset) were investigated and compared with 185 healthy peers. APC- resistance (FV:Q506), protein C, protein S, antithrombin, heparin cofactor II (HCII), histidine-rich glycoprotein (HRGP), and prothrombin (F.II), factor XII (F.XII), plasminogen, homocysteine and lipoprotein (a) (Lp(a)) were investigated. In 59% of patients investigated one thrombotic defect was diagnosed, 19.6% showed two thrombotic risk factors, while in 21.4% of children investigated no risk factor could be identified. Single defects comprised established causes of inherited thrombophilia: FV:Q506 (homozygous n = 10, heterozygous n = 69), protein C (homozygous n = 1; heterozygous n = 31), heterozygous type I deficiency states of protein S (n = 7), antithrombin (n = 7) and homocystinuria (n = 6); potentially inherited clotting abnormalities which may be associated with thrombophilia: F.XII (n = 3), plasminogen (n = 2), HCII (n = 1), increased HRGP (n = 4); new candidate risk factors for thrombophilia: elevated plasma levels of Lp(a) (n = 26), F.II (n = 1). Heterozygous FV:Q506 was found in combination with heterozygous type I deficiency states of protein C (n = 2), protein S (n = 13), antithrombin (n = 8) and HCII (n = 1), increased Lp(a) (n = 13), and once each with elevated levels of F.II, moderate hyperhomocysteinemia, fibrinogen concentrations >700 mg/dl and increased HRGP. In addition to the association with FV:Q506, heterozygous protein C type I deficiency was combined with deficiencies of protein S (n = 2), antithrombin (n = 1), and increased Lp(a) (n = 3). One patient showed protein C deficiency along with familially increased von Willebrand factor >250%. Besides coexistence with FV:Q506 and protein C deficiency, protein S deficiency was combined with decreased F.XII and increased Lp(a) in one subject each. Furthermore, we found combinations of antithrombin deficiency/elevated Lp(a), hyperhomocysteinemia/Lp(a), deficiency of HCII/plasminogen, and plasminogen deficiency along with increased Lp(a) each in one. Increased prothrombin levels were associated with fibrinogen concentrations >700 mg/dl and with HCII deficiency in one child each. Carrier frequencies of single and combined defects were significantly higher in patients compared with the controls. Conclusion In conclusion, data of this multicentre evaluation indicate that paediatric thromboembolism should be viewed as a multifactorial disorder.

The course of ADAMTS‐13 activity and inhibitor titre in the treatment of thrombotic thrombocytopenic purpura with plasma exchange and vincristine

The therapeutic efficacy of plasma exchange (PE) in thrombotic thrombocytopenic purpura (TTP) is attributed to the restoration in ADAMTS‐13 (a disintegrin and metalloproteinase with thrombospondin motif‐13) activity by substitution of the enzyme and removal of ADAMTS‐13‐neutralizing autoantibodies. We explored this rationale by analysing ADAMTS‐13 activity and corresponding inhibitor levels during PE‐treatment in 27 episodes from 23 adults with TTP. All patients with an initial episode of TTP (n = 14) and nine of 11 patients with a relapse showed severe ADAMTS‐13 deficiency. ADAMTS‐13 inhibitors were detected in 81% of these patients. Twenty‐one patients responded to PE‐therapy and two patients died. For patients with severe ADAMTS‐13 deficiency, 15 patients (71%) showed a PE‐induced recovery in ADAMTS‐13 activity and six patients (29%) had persistent severe ADAMTS‐13 deficiency despite clinical response. Three patients with recurrent TTP demonstrated a permanent increase in inhibitor titre during therapy. Six patients (43%) with an initial episode of TTP displayed a transient increase in inhibitor titre during PE‐therapy, which was associated with deterioration in clinical and haematological symptoms of TTP. Treatment with vincristine induced an immediate increase in platelet count and ADAMTS‐13 activity in seven of eight patients. We conclude that ADAMTS‐13 activity and inhibitor levels, as measured using current methodology, do not solely determine the clinical course of TTP.

Evaluation and clinical application of a new method for measuring activity of von Willebrand factor-cleaving metalloprotease (ADAMTS13).

Thrombotic thrombocytopenic purpura (TTP) is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFcp). Based on partial amino acid sequence and genome-wide linkage analysis of pedigrees with congenital TTP, VWFcp was recently identified as a new member of the ADAMTS family and designated ADAMTS13. We developed a new, rapid, and simple method for measuring VWFcp activity based on the positive correlation between VWF multimeric size and Ristocetin cofactor activity (VWF:RCo). After dilution of plasma with low ionic Tris buffer and activation of the protease with barium chloride, a VWF concentrate is digested in the presence of urea. Subsequently, the residual VWF:RCo of the samples is assessed and used to calculate the VWFcp activity of the samples. The accuracy of the new technique is verified by estimating VWFcp activity for 282 plasma samples with the RCo-based assay and the original immunoblotting assay. The method is reproducible as shown by low intra- and interassay coefficients of variation (2.8% and 7.5% for normal samples, respectively, and 8.7% and 12.9% for abnormal samples, respectively). Furthermore, the clinical application of the new method is illustrated by measuring VWFcp of 14 patients with 22 episodes of acute TTP as well as other thrombotic, thrombocytopenic, or hemolytic disorders. Severe VWFcp deficiency was restricted to patients with acute, classic TTP. The majority of patients with low titer inhibitor respond to plasma exchange treatment with increase of VWFcp activity, whereas VWFcp deficiency persists in some patients with high titer inhibitor despite clinical remission.

Prothrombotic risk factors in childhood stroke and venous thrombosis.

Abstract Many studies have shown a high percentage of venous thromboses in children to be associated with haematological disorders. However, studies assessing the influence of haemostaseological disorders on paediatric stroke are rare. We compared 26 children with cerebral infarction (median age 2 months, range 0–16.2 years) and 17 with venous thrombosis (median age 4.5 years, range 0–17 years) with regard to prothrombotic risk factors. Prothrombotic disorders were found in 8 out of 26 patients with cerebral infarction (FV Leiden mutation: n = 4; protein C deficiency: n = 1; FV Leiden mutation + protein C deficiency: n = 2; prothrombin mutation G20210A: n = 1) and in 13 out of 17 with venous thrombosis (FV Leiden mutation n = 3; protein C deficiency n = 5; elevated HRGP + PAI: n = 1; combined deficiency of AT, protein C and plasminogen: n = 1; F XII deficiency: n = 1; lupus anticoagulans n = 1; FV Leiden + F XII deficiency + lupus anticoagulans + PAI: n = 1). Comparison of these prevalences with those of 150 healthy paediatric controls showed in children with FV Leiden mutation and/or protein C deficiency an increased risk of cerebral infarction (patients vs. controls: 26.9% vs. 6%; OR 5.77; 95%-CI 1.92–17.3; P = 0.0031) as well as of venous thrombosis (53% vs. 5.3% 19.9; 95%-CI 6–65.6; P < 0.0001). This result is in contrast with reports on thrombophilia in cerebral infarction in adult patients. Conclusion Our results indicate that FV Leiden mutation and protein C deficiency may contribute to the multifactorial aetiology of stroke in early childhood.

Valproate Therapy Induces von Willebrand Disease Type I

Summary: To investigate the increased tendency of hemorrhage in patients receiving valproate (VPA) therapy, we studied coagulation parameters in 30 randomized children of a group of 83 children receiving antiepileptic drug (AED) therapy. Besides a reduction in fibrinogen concentration and platelet count, we observed a significant decrease in factor VIII‐complex. A decrease in factor VIII:C was noted in 33%, a decrease in von Willebrand factor (vWF:Ag) was noted in 83% and a decrease in ristocetin‐cofxtor activity (vWF:Rcof) was noted in 66% of the children. We classified a von Willebrand syndrome type I in 67% of our patients receiving VPA therapy. Sixty‐three percent of patients had a history of bleeding, and 23% had a prolonged bleeding time. We compared our results with those of a control group and of a group of patients with congenital von Willebrand disease (vWD), from which patients with multimer types II and III were excluded. Because coagulation parameters in patients with congenital vWD are similar to those receiving AED therapy, we designated the increased tendency to hemorrhage as VPA‐induced vWD. The decrease in coagulation parameters were not dependent on either VPA dose or period of administration. In patients receiving VPA therapy, this result must be considered, especially during surgical intervention and after traumatic events.