Intravascular inhibition of factor VIIa and the analogue NN1731 by antithrombin.

NN1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa- and FVIIa-AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t(½) ≈1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t(½) ≈0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t(½) ≈1·1 h) response. Western Blot and FVIIa-AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue.

Factor VIII mutations in 42 Moldovan haemophilia A families, including 12 that are novel.

Haemophilia A (HA) is a bleeding disorder caused by mutations within the X-linked F8 gene. A series of 42 unrelated Moldovan patients with HA had their disease-causative mutation determined to provide clinically valuable genotyping information for a historically underserved population and to utilize factor VIII (FVIII) structural information to analyse the effects of haemophilic missense substitutions. DNA samples were analysed to detect intron 22 and intron 1 inversions followed by heteroduplex analysis of PCR-amplified fragments containing all exonic sequences. Missense sites identified by DNA sequencing were visualized in the recently described crystal structures of human FVIII. Of the 26 different point mutations, 12 were novel. Gel electrophoresis identified samples with a second major DNA band that migrated abnormally; these amplified products were sequenced. Thirteen intron 22 inversions and one intron 1 inversion were found. Two patients had large, partial gene deletions and there were six frameshift, two non-sense, two splicing and 16 missense genotypes. Two subjects with an intron 22 inversion and one with a large, partial gene deletion developed an alloimmune inhibitor. Their localization suggests intra- and possibly inter-molecular interactions that are important for the structural integrity and/or procoagulant function of FVIII.

Monitoring inhibitor patients with the right assays.

The inhibitor titer is the most important clinical measurement in inhibitor patients, and the Nijmegen method is preferable to the original and well-established Bethesda assay for this purpose; however, both methods have high inter-laboratory variability. Monitoring inhibitor patients after treatment with bypassing agents is difficult. Treatment with recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) can be monitored with assays for FVII clotting activity or with a specific assay for FVIIa; while the latter is more reproducible, its relevance to the clinical response of individual patients remains unclear. Recent years have also witnessed a revival of global assays, which contribute useful additional information and which may be more relevant to the hemostatic state of individual inhibitor patients. One such assay currently undergoing a renaissance is the thrombin generation test (TGT). The TGT has many methodological variations--several of which have been studied in our laboratory--and shows the most promise for use in treatment monitoring. This article reviews the assays most appropriate for monitoring inhibitor patients and discusses some of the most recent developments in the use of global assays in this indication.

Arthropathy in inhibitor patients: differences in the joint status.

Inhibitor development represents the most significant complication of hemophilia treatment today. Although it has long been established that hemophilia patients with inhibitors face a higher risk (and a greater extent) of joint morbidity than their non-inhibitor counterparts, there remains a paucity of scientific data supporting this observation. Several issues need to be resolved before the risk of greater joint damage in inhibitor patients can be verified. This article explores these issues in an attempt to confirm and explain the differential joint morbidity observed between inhibitor and non-inhibitor patients. However, there remain significant gaps in our knowledge of the biology of inhibitor development, and greater understanding in this area will produce more effective therapies that might ultimately prevent or attenuate their development altogether.

Inhibitor development in hemophiliacs: the roles of genetic versus environmental factors.

Approximately 5 to 7% of patients with hemophilia A have inhibitory antibodies to factor (F) VIII, which increases to approximately 13% in patients with severe disease. The strongest determinant of the risk of inhibitor development identified is the type of mutation in the FVIII gene that gives rise to the disease. However, accumulating evidence clearly indicates that other genetic factors (e.g., major histocompatibility complex alleles and other immune-modulatory genes) and factors associated with treatment (e.g., type of FVIII concentrate, route of administration, and age of first exposure) may also influence the risk of inhibitor development. There is much interest in identifying such genetic and treatment-related factors to help minimize the risk of inhibitor development and improve treatment outcomes.

Inhibition of coagulation by macromolecular complexes.

The role of vertebrate blood coagulation is to rapidly prevent the loss of body fluids following vascular injury without compromising blood flow through either the uninjured or damaged vessels. To achieve this the coagulation network is initiated and regulated by a complex network of interactions that are under the control of both positive and negative feedback loops that result in controlled fibrin deposition and platelet activation only at the site of injury. Anticoagulant molecules play key roles in preventing inappropriate initiation of coagulation as well as down-regulating thrombin generation at the site of injury. Tissue factor pathway inhibitor (TFPI) inhibits the initiation complex, antithrombin (AT) inhibits the active serine proteases directly, whereas the activated protein C pathway inhibits coagulation by inactivating the cofactors V and VIII. In this review the structure and function of these anticoagulant molecules and their inhibitory complexes is discussed.

Development of selected coagulation factors and anticoagulants in preterm infants by the age of six months.

The development of the coagulation and anticoagulation system in preterm infants was assessed, with special emphasis on extremely low birth weight (ELBW) infants and haemorrhagic or other complications after birth. Coagulation factors II (prothrombin), V (FV), VII (FVII) and X (FX) were analysed at birth and at a corrected age of six months. In addition, antithrombin (AT), protein C (PC) and protein S (PS) were measured at six months, and DNA samples were tested for Factor V Leiden (R506Q). Eighty-two infants, with a median gestational age (GA) of 32 weeks (range 24-36) and a median birth weight of 1562 g (range 695-3520), were studied. Fifteen of these were ELBW infants (range 695-1000g). Prothrombin, FV, FVII and FX reached healthy term six-month-old infant activity levels. Prothrombin and FX did not reach adult values; median activity levels remained at 82% and 78%, respectively. During the follow up, the FV and FVII levels of the ELBW infants (GA 24-27 weeks) increased more than those of the preterm infants born with higher GA (p < 0.001). At birth, prothrombin correlated significantly with FV, FVII and FX (p < 0.001). FVII at birth and at six months correlated significantly with PC (p = 0.021 and p = 0.009, respectively). These findings indicate that the gain in the coagulation factor concentrations in infancy is greatest in infants with the lowest GA at birth. Interesting new inter-relations of coagulation factor and physiological anticoagulant levels may indicate that there are still unrecognised pathways in the function of newborn haemostasis.

Embryogenesis and gene targeting of coagulation factors in mice.

Genetic or acquired thrombophilia of the pregnant mother has been associated with the occurrence of gestational vascular disease and recurrent fetal loss and may contribute to the aetiology of pre-eclampsia. This chapter reviews insights into this link between thrombophilia and pregnancy complications that were gained from the study of genetically altered mice. These studies strongly support the notion of a cause-effect relationship between altered function of the thrombomodulin-protein C pathway and adverse pregnancy outcome. Analysis of the mouse models highlights unique aspects of vascular structure and function at the feto-maternal interface, and exposes new biological functions of natural anticoagulant pathways in pregnancy. These roles are unrelated to the maintenance of vascular patency and may be mediated through specific signalling pathways activated by coagulation factors. Abnormal signalling by placental trophoblasts at the feto-maternal interface is suggested as a hitherto unrecognized mechanism that may underlie adverse pregnancy outcome associated with haemostatic disorders.

The dynamics of thrombin formation.

The central event of the hemostatic process is the generation of thrombin through the tissue factor pathway. This is a highly regulated, dynamic process in which thrombin itself plays many roles, positively and negatively its production and destruction. The hemostatic process is essential to normal physiology and is also the Achilles heel of our aging population. The inappropriate generation of thrombin may lead to vascular occlusion with the consequence of myocardial infarction, stroke, pulmonary embolism, or venous thrombosis. In this review, we summarize our present views regarding the tissue factor pathway by which thrombin is generated and the roles played by extrinsic and intrinsic factor Xa generating complexes in hemostasis and the roles of the stoichiometric and dynamic inhibitors that regulate thrombin generation.

Platelets and anticoagulant capacity in patients with inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) are susceptible to thromboembolic complications. Several mechanisms can be responsible, including abnormal regulation of coagulation activity, disturbances of fibrinolysis, inflammatory reactions and thrombocytosis. The aim of this study was to assess hemostatic alterations in these parameters during exacerbation of disease. We studied disease activity in 99 IBD patients receiving anti-inflammatory therapy, in relation to: procoagulant markers, i.e. prothrombin fragment F1 + 2 (F1 + 2), D-dimer and platelet count, anticoagulant markers, i.e. protein C, protein S and antithrombin, and a mediator of inflammation (IL-6). Coagulation activity and platelet count were increased during active disease in IBD patients compared with those in a state of remission. The IL-6 concentrations were positively correlated with disease activity and thrombocytosis in patients with ulcerative colitis, but no association with the anticoagulant capacity could be demonstrated except for a decrease in protein C during high disease activity.

Pathobiology of pulmonary hypertension. The role of platelets and thrombosis.

With the rare exceptions of PAH associated with antiphospholipid antibodies, genetic platelet dysfunction, or inherited deficiencies of antithrombotic pathways, the thrombotic lesions are secondary, but frequently occurring, in most cases of primary or secondary PAH. Pulmonary arterial hypertension is associated with thrombotic lesions and persistent vasoconstriction and structural remodeling of PA. Activated platelets interact with the PA wall and may contribute to the functional and structural alterations of pulmonary vessels by releasing vasoactive factors and mitogenic mediators.

Relaciones entre la estructura molecular-función del FVIII y el mecanismo de acción de los inhibidores anti-FVIII en hemofilia A / Relationship between molecular structure and function of factor VIII and mechanism of action of anti-FVIII inhibitors in hemophilia A

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Evaluation of the hypercoagulable state. Whom to screen, how to test and treat.

Venous thromboembolism is a common disease that causes significant morbidity and mortality. In recent years, the ability to diagnose inherited genetic defects and common acquired conditions predisposing to thrombosis has greatly increased. Venous thromboembolism is now understood to be a complex interaction of genetic and environmental factors leading to thrombosis. Integrating the various factors to individually assess thrombotic risk still poses a challenging clinical problem that will likely become easier as more data accumulate. As the ability to accurately assess risk increases, the data can then be translated into tailored treatment regimens. Until then, only general guidelines regarding evaluation and management are available. In the future, it is likely that other prothrombotic conditions will be elucidated, adding to the pool of data.

The vitamin K-dependent proteins: an update.

Historically known for its role in blood coagulation, vitamin K also has been shown to be required for the physiologic activation of numerous proteins that are not involved in hemostasis. Over the last 20 years, vitamin K-dependent proteins have been isolated in bone, cartilage, kidney, atheromatous plaque, and numerous soft tissues. Although the precise mechanism of action of many of these proteins remains to be determined, their discovery has proven important from a physiologic point of view.

[Assessment of the hemostatic system in healthy preterm and fullterm newborns]. / Valoración del sistema hemostático en el recién nacido pretérmino y a término sano.

Assessment of the hemostatic system in premature and newborn infants. When the newborn is compared to older children and adults, several differences in the coagulation and fibrinolytic systems have been described; near-adult values are achieved for most components by 6 months of life. The newborn has an impaired platelet aggregation, reduced synthesis of clotting factors and inhibitors and molecular abnormalities in some proteins of blood coagulation. The role of fibrinolysis is, however, less well known. We have performed a study to assess the fibrinolytic activity in 100 healthy newborns and we have found a marked increase of the fibrinolytic activity, mainly related to an enhancement of plasminogen activators (t-PA) and reduction of fibrinolysis inhibitors, without systemic fibrinolysis. These results would suggest that the fibrinolytic system in the newborn is in equilibrium with the clotting system in order to preserve physiological hemostasis.