Ruediger E. Scharf

Genetic typing of human platelet antigen 1 (HPA‐1) by oligonucleotide ligation assay in a specific and reliable semi‐automated system

Genotyping of platelet alloantigens with the possibility of using any type of cellular material as a source of DNA has become a preferred procedure, particularly in thrombocytopenic patients when platelet counts are too low for phenotyping. Recently human platelet antigen 1 (HPA‐1) has been identified as an inherited risk factor for coronary thrombosis. The different detection methods currently used have disadvantages for large‐scale DNA diagnosis, including the need for electrophoresis (allele‐specific restriction enzyme analysis, amplification with sequence‐specific primers) or the potential risk of reduced specificity (allele‐specific oligonucleotide hybridization). In this report we describe the adaptation of an automated oligonucleotide ligation assay to genotype HPA‐1 in polymerase chain reaction (PCR)‐amplified DNA samples. HPA‐1a and HPA‐1b phenotypes corresponded to the results of the different genotyping assays. The genotypes determined with the ELISA‐based PCR‐oligonucleotide ligation assay were in 100% concordance with the results obtained by conventional allele‐specific restriction enzyme site analysis and PCR amplification with sequence‐specific primers. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to genotype human platelet antigens that can rapidly be applied to large population screening.

Transforming growth factor-β1 enhances the antifibrinolytic and prothrombotic state of growing endothelial cells in a cell cycle-specific manner

Cell cycle‐dependent modulation of protein expression may influence the balance of antithrombotic and prothrombotic properties of endothelial cells. In the present study, we examined the regulation of prothrombotic and antithrombotic molecules by transforming growth factor‐β1 (TGF‐β1) during distinct phases of the cell cycle in human umbilical vein endothelial cells. In the absence of TGF‐β1, the expression of thrombomodulin, the plasminogen activators u‐PA and t‐PA, and their inhibitor PAI‐1 was significantly increased in the S/G2 compared to the G1 phase. Treatment of endothelial cells with TGF‐β1, however, resulted in elevated expression of PAI‐l specifically in the S/G2 phase, while t‐PA and u‐PA increased to the same extent in both the Gl and S/G2 phase. These findings demonstrate that the expression of a subset of hemostatically relevant proteins is regulated during endothelial cell cycle and that TGF‐β1 can differentially modulate cell cycle‐controlled protein expression.—Stoldt, V. R., Schnorr, O., Schulze‐Osthoff, K., Scharf, R. E. Transforming growth factor‐β1 enhances the antifibrinolytic and prothrombotic state of growing endothelial cells in a cell cycle‐specific manner. FASEB J. 20, E55–E60 (2006)

Highly efficient lentiviral transduction of phenotypically and genotypically characterized endothelial progenitor cells from adult peripheral blood

Postnatal vasculogenesis has been implicated as an important mechanism for neovascularization via bone marrow-derived endothelial progenitor cells (EPCs) circulating in peripheral blood. In preparation of the utilization of EPCs in clinical protocols, we have generated blood-derived EPCs according to two established protocols by culturing either nonadherent mononuclear cells on fibronectin or adherent mononuclear cells on collagen. To explore the feasibility of these EPCs for their potential clinical use as target cells for genetic transduction to enhance their thromboresistance, newly designed retroviral and lentiviral gene ontology expression vectors were tested. Whereas cell clusters derived from the nonadherent cells demonstrated an only limited proliferative potential, cell colonies derived from collagen-adherent cells expanded more than a million-fold. Characterization of the exponentially growing cells by surface antigen and gene expression profiling revealed a consistently strong expression of characteristic endothelial markers, whereas expression of leukocyte markers was gradually lost. Using a single-step transduction protocol, we were able to achieve gene transfer efficiency of up to 99%. Our results suggest that the generated blood-derived EPC population might be attractive target cells for tissue engineering and gene therapy protocols due to their well defined phenotype, extensive proliferative potential, and efficient genetic transducibility, three important qualities that need to be defined prior to any clinical use.

Interdisciplinary management of total knee replacement in a haemophilia patient with high‐titre inhibitor and severe arthropathy complicated by an aneurysmatic bone cyst

Until recently, surgical intervention for haemophilia patients with inhibitors, particularly those with highresponding inhibitors, was limited to emergency treatment of lifeor limb-threatening clinical conditions. The difference in treatment compared with haemophilia patients without inhibitors was owing to the lack of haemostatic agents that could be used to cover perioperative procedures in inhibitor patients. The availability of effective alternative haemostatic agents such as recombinant factor VIIa (rFVIIa, NovoSeven) was a precondition that surgical interventions such as joint replacement have become a clinical option for all haemophilia patients regardless of their inhibitor [1–3]. There are different treatment strategies for performing surgical procedures in inhibitor patients [4]: in low-responding patients, when the inhibitor titre is <5 Bethesda Units (BU), it is generally enough to administer a high dose of clotting factor concentrate. In high-responding patients, with no inhibitor at the time of surgery, the surgical procedure can be performed administering the missing clotting factor. After anamnestic response with high-titre elevation, it is necessary to use bypass agents such as rFVIIa (NovoSeven). Here in, we report on the interdisciplinary management of a two-stage surgical procedure for a total knee joint arthroplasty (TKA) in a haemophilic patient with anamnestic high-titre inhibitor. A 35-year-old male patient with severe haemophilia A developed a high-titre inhibitor (112 BU) at the age of 26 years after more than 200 exposure days to a recombinant factor VIII concentrate. The genetic defect causing haemophilia was an intron 22 inversion of the factor VIII gene. As the patient rejected immune tolerance therapy, on-demand treatment with rFVIIa was initiated. He now presented with acute pain in his left distal femur and being unable to bear weight. X-rays revealed an increasing osteolytic cystic bone tumour, severe knee arthropathy and a large pseudotumour posteriorly (Fig. 1). He had experienced recurrent bleeds into this bone tumour of the distal femur owing to a bone tumour-related infraction of the cortical bone. A two-stage procedure was favoured: initially a biopsy of the distal femur was taken confirming the diagnosis of an active aneurysmatic bone cyst. This surgical procedure was covered with rFVIIa (100 lg kg body weight (bw)) in order to avoid anamnestic response and therefore not to booster inhibitory antibody to factor VIII:C. Preoperatively, during surgery and postoperatively he received bolus injections of rFVIIa (100 lg kg bw) every 6 h for 7 days. Because of the osteolytic bone tumour of the distal femur with bony instability and concomitant severe knee arthropathy we decided to resect the bone tumor and implant custom-made constrained bicondylar TKA prosthesis, 2 weeks after the first operative procedure. Surgery was performed through a standard approach under tourniquet control, implanting a cementless fully constrained tumour knee prosthesis, including synovectomy, removal of a large pseudotumour and soft-tissue balancing with release of quadriceps (Fig. 2). He preoperatively measured the value of the inhibitor antibody to factor VIII:C to be below the detection limit, therefore surgery was covered perioperatively by recombinant factor VIII replacement therapy by episodic bolus injections 40 IU kg bw every 6 h for 3 days, followed by Correspondence: Ansgar Ilg, MD, Department of Orthopaedics, University of Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany. Tel.: 0049–211-811 6688; fax: 0049–211-811 7073; e-mails: ilg@med.uni-duesseldorf.de; ansgarilg@gmx.net

The G1691A Mutation of the Factor V Gene (Factor V Leiden) and the G20210A Mutation of the Prothrombin Gene as Risk Factors in Thrombotic Microangiopathies

Factor V Leiden (FVL) mutation and prothrombin G20210A mutation are common hereditary risk factors for venous thrombosis. In the current study, 40 patients (mean age ± standard deviation, 35 ± 11 years) and 764 healthy control subjects (mean age ± standard deviation, 37 ± 14 years) were enrolled to assess the potential role of these mutations in the manifestation of thrombotic microangiopathies. Compared with controls, neither the heterozygous FVL mutation (7.5% vs 8.5%; P = 1) nor the heterozygous prothrombin mutation (2.5% vs 2.8%; P = 1) was more prevalent in the patients. The findings do not support a significant role of FVL and prothrombin mutations as risk factors for the manifestation of thrombotic microangiopathies. Thus, screening for these mutations does not allow the identification of individuals at increased risk for these rare thrombotic disorders.

Recombinant Factor VIIa for the Prophylaxis of Perioperative Hemorrhage in a Patient With Congenital Factor XI Deficiency Undergoing Brain Tumor Neurosurgery

The authors report on the first successful use of recombinant activated factor VII for the prophylaxis of bleeding during brain tumor neurosurgery in a patient suffering from inherited factor XI deficiency. Using the agent, surgery was performed without any bleeding complications. In this setting, off-label use of recombinant activated factor VII appears to be a promising alternative for patients suffering from this rare hemostatic defect with hitherto limited therapeutic options.