Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three types--type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype characteristics of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD. One hundred fourteen patients belonging to 78 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF . Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The impact of novel candidate missense mutations and potential splice site mutations was predicted by in silico assessments. We identified mutations in 66 index patients (IPs) (84.6%). Mutation detection rate was 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected comprising 37 missense mutations (54.4%), 10 small deletions (14.7%), two small insertions (2.9%), seven nonsense mutations (10.3%), five splice-site mutations (7.4%), six large deletions (8.8%) and one silent mutation (1.5%). Twenty-six of these mutations were novel. Furthermore, in type 1 and type 2 VWD, the majority of identified mutations (74% vs. 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). Genotyping in VWD is a helpful tool to further elucidate the pathogenesis of VWD and to establish the relationship between genotype and phenotype.

Summary. The PFA-100Ò (PFA) diagnostic system for the detection of platelet dysfunction was evaluated to determine reference ranges in a normal population. The PFA determines the primary haemostasis capacity (PHC) of anticoagulated whole blood, expressed by the system's closure time (CT). In this study the CT reference ranges were determined for blood samples collected in 105 mmol/l (3´2%) buffered citrate and the effect of gender, smoking, and use of oral contraceptives on reference ranges was assessed. Each of the 309 healthy blood donors from ®ve blood centres was con®rmed to have normal platelet function before inclusion in the study. Blood samples were tested in duplicate with both the collagen/epinephrine (Col/Epi) and collagen/ADP (Col/ADP) test cartridges.PFA reference ranges (90% central intervals of measured closure times) for both cartridge types were similar for all groups. Subgroup analysis showed that neither gender nor oral contraceptive usage had any effect on PHC. The 95% cut-off value for the Col/Epi CT was slightly higher for smokers than for non-smokers, an effect more pronounced in female than in male donors. However, the small difference did not justify establishment of speci®c reference ranges for smokers. Data from all included subjects were pooled to calculate the CT reference ranges for blood samples collected in 105 mmol/l buffered citrate (Col/Epi 82±150 s; Col/ADP 62±100 s). Normal levels of ®brinogen, as well as normal platelet counts and normal haematocrit levels, appeared not to in¯uence the PHC. Because slight but signi®cant differences of the reference ranges were observed between some of the participating sites, in-house con®rmation of these reference range guidelines is recommended.

Summary. Bleeding complications are the most common and unwanted side-effect of oral anticoagulant therapy. We report three patients in whom mutations in the factor IX (FIX) propeptide were found to cause severe bleeding during coumarin therapy. Strikingly, the bleeding occurred within the therapeutic ranges of the prothrombin time (PT) and international normalized ratio (INR). In all three patients coumarin therapy caused an unusually selective decrease of FIX activity (FIX:C) to levels below 1-3%. Upon withdrawal of coumarin, FIX:C increased to subnormal or normal values of 55%, 85% and 125%, respectively. Analysis of the FIX gene revealed two different missense mutations affecting the Ala-10 residue in the propeptide coding region: Ala[GCC] to Val [GTC] in two patients and Ala[GCC] to Thr[ACC] in one patient. No further mutation was detected by screening 195 random blood donors for mutations at Ala-10, thus excluding a frequent polymorphism at this position. The mutation in the FIX propeptide at a position which is essential for the carboxylase recognition site causes a reduced affinity of the carboxylase enzyme to the propeptide. This effect leads to an impaired carboxylase epoxidase reaction which is decisively triggered by the vitamin K concentration. Determination of FIX and APTT in addition to PT and INR is therefore recommended in coumarintreated patients with an uncommon bleeding pattern.

Objective: Little is known about the influence of preterm delivery and perinatal risk factors on development and expression of the coagulation system in extremely preterm infants. The objective of this study was to determine reference values for the components of the coagulation system at the first day of life in extremely preterm infants.Study Design: Components of the coagulation system were examined retrospectively in 132 extremely preterm infants. Patients were grouped according to clinical criteria for preterm delivery: group A: maternal indication; group B: uteroplacental dysfunction; group C: systemic inflammation.Result: Levels of coagulation factors VII and X rose with increasing gestational age, whereas fibrinogen and coagulation factors II, V and VIII remained constant. Levels of factors V and VIII were higher than those of vitamin K-dependent factors. If preterm delivery was caused by placental disorder (group B) or chorioamnionitis (group C), levels of factor II, VIII and X were significantly lower, whereas factor V and VII levels did not differ. In group C fibrinogen levels in group C were higher compared with group A. Conclusion:Identification of perinatal risk factors may help to define patients at risk of bleeding disorders.

On-pump cardiac surgery is accompanied by complex alterations of hemostasis. The excessive postoperative bleeding has been attributed to acquired platelet dysfunction, impaired plasmatic coagulation, and increased fibrinolysis. The characterization of the hemostatic defects responsible for bleeding is crucial for specific treatment and optimal clinical management of the patient. For rapid determination of platelet-dependent primary hemostatic capacity (PHC), the Platelet Function Analyzer PFA-100 system is available. To evaluate the PFA performance in perioperative monitoring, a study was performed in 49 patients selected for low bleeding risk undergoing selective primary coronary artery bypass grafting (CABG). We compared PHC with Simplate bleeding time (BT) and platelet aggregometry. Furthermore, we analyzed global hemostasis by thromboelastography (TEG) and plasmatic coagulation by standard clotting tests prothrombin time (PT, Quick), activated partial thromboplastin time (aPTT), thrombin time (TT) and clotting factors and fibrinolysis by batroxobin (reptilase) time (RT). In all patients BT was postoperatively increased by 1.5- to 2-fold irrespective of perioperative complications and decreased to mildly prolonged values on the first postoperative day (1st day). In patients without complications, PHC in both collagen-adenosine diphosphate closure time (CADP-CT: 83 seconds preop, 78 seconds postop, and 74 seconds 1st day) and collagen-epinephrine closure time (CEPI-CT: 98 seconds preop, 95 seconds postop, 85 seconds 1st day) remained nearly stable. Apart from a patient with postoperative moderate thrombocytopenia, in bleeding patients no other significant defect of postoperative platelet hemostatic capacity was observed. However, on 1st day, the PHC of those patients was significantly reduced compared with non-bleeding patients. In patients with postoperative myocardial ischemia, increased PHC was identified by significantly shorter postoperative CADP-CT (66 seconds vs. 83 seconds) than in uncomplicated patients. By aggregometry, partial platelet dysfunction was observed in some patients without correlation to bleeding complications. In seven of 9 patients the postoperative bleeding complication was attributed to prolonged heparin anticoagulation and/or mildly enhanced fibrinogenolysis/fibrinolysis by TEG and standard plasmatic coagulation tests (TEG: k time 18 minutes vs. 8 minutes; aPTT: 47 seconds vs. 32 seconds; TT: 18.0 seconds vs. 12.3 seconds) and (RT: 19.5 seconds vs. 17.7 seconds). The impairment of PHC, platelet aggregation, and clotting factors observed on the 1st day in bleeding and in intra-aortic balloon pump (IABP) patients are most likely secondary effects, for example, loss of active platelets and clotting factors, to the primary postoperative bleeding or implantation of the IABP. In conclusion, our data indicate that in standard CABG procedures highly variable alterations of the hemostatic system occur after cardiopulmonary bypass (CPB) even in patients with assumed low operative risks. For id...