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PubMedDate not supplied

Hemophilic dogs response to Bridge anticoagulant neutralizing agent.

When Bridge anticoagulant neutralizing agent, prepared by column fractionation, was infused with factor VIII concentrate into a hemophilic dog, the apparent level of circulating factor VIII was markedly increased. Material isolated from 30 ml donor plasma raised the level of factor VIII nearly three-fold, during the first post-transfusion hour. This was achieved without shortening the period during which a hemostatically-effective level of factor VIII was maintained. The same dose also reduced the amount of factor VIII required for prompt and successful hemostasis after standardized template gingival biopsy.

Animals↗
PubMedDate not supplied

The response of an acquired Factor V inhibitor to activated Factor IX concentrate.

A 91-year-old man developed a severe bleeding diathesis postoperatively. Laboratory studies showed an inhibitor to factor V which was identified as IgG. The patient failed to respond to fresh-frozen plasma and platelet transfusions, but demonstrated both clinical and laboratory improvement after transfusion with an activated prothrombin complex concentrate (Autoplex). Patients with refractory inhibitors to factors VIII or IX have been managed successfully with this concentrate; however, this case demonstrates that is also may be of value in managing patients with refractory inhibitors to factor V.

Aged↗
PubMed2003

Molecular mechanisms of mild and moderate hemophilia A.

Mutations responsible for mild/moderate hemophilia A were extensively characterized over the last 15 years and more than 200 mutations have been identified. However, most of the molecular mechanisms responsible for the reduced factor (F)VIII levels in patients' plasma were determined only recently. Recent progresses in the study of the FVIII molecule three-dimensional structure provided a major insight for understanding molecular events leading to mild/moderate hemophilia A. This allowed prediction of mutations impairing FVIII folding and intracellular processing, which result in reduced FVIII secretion. Mutations potentially slowing down FVIII activation by thrombin were also identified. A number of mutations were also predicted to result in altered stability of activated FVIII. Biochemical analyses allowed identification of mutations reducing FVIII production. Mutations impairing FVIII stability in plasma, by reducing FVIII binding to von Willebrand factor (VWF) were also characterized. Defects in FVIII activity, notably slow activation by thrombin, or abnormal interaction with FIXa, were also recently demonstrated. Biochemical analysis of FVIII variants provided information regarding the structure/function relationship of the FVIII molecule and validated predictions of the three-dimensional structure of the molecule. These observations also contributed to explain the discrepant activities recorded for some FVIII variants using different types of FVIII assays. Altogether, the study of the biochemical properties of FVIII variants and the evaluation of the effects of mutations in three-dimensional models of FVIII identified molecular mechanisms potentially explaining reduced FVIII levels for a majority of patients with mild/moderate hemophilia A. It is expected that these studies will improve diagnosis and treatment of this disease.

Factor IXa↗
PubMed2003

Thirty-four novel mutations detected in factor VIII gene by multiplex CSGE: modeling of 13 novel amino acid substitutions.

Detection of causal mutations is required for genetic counseling. Molecular modeling combined with patients' phenotype provides significant insight into structure-function relationship of factor (F)VIII molecule. Our objective was to identify defects in the gene of FVIII by a sensitive and simple scanning technique with high throughput in order to study molecular mechanisms by which novel amino acid substitutions may lead to hemophilia A. A cohort of 81 families with mild, moderate and severe hemophilia A negative in intron 22 inversion was studied. For detection of mutations in the FVIII gene a conformation sensitive gel electrophoresis (CSGE) was modified by multiplexing. Thirteen novel amino acid substitutions were studied by molecular modeling and a correlation with the cross-reactive material (CRM) phenotype was performed. In 74 families, 59 different mutations were detected. Six different mutations were recurrent in 21 unrelated families. Thirty-four novel mutations included 19 point mutations, four small insertions, nine small deletions and two complex mutations. Thirteen novel amino acid substitutions occurred at residues conserved in FVIII orthologs. Five of them were associated with a discrepancy between FVIII activity and antigen; another five with CRM reduced phenotype and one with undetectable FVIII antigen. Multiplexing of the CSGE significantly increased its throughput without substantial loss of sensitivity. Molecular modeling suggested mechanisms by which substitutions at residues 382 and 569, located outside the proposed FIXa-binding region, may influence FVIII/FIXa interaction. His2155 was predicted to participate in FVIII/VFW binding.

Amino Acid Substitution↗
PubMed2003

Functional analysis of the EGF-like domain mutations Pro55Ser and Pro55Leu, which cause mild hemophilia B.

We studied the functional role of two mutations, Pro55Ser and Pro55Leu, located in the N-terminal Epidermal Growth Factor-like domain (EGF1) of coagulation factor (F) IX. Both mutations cause mild hemophilia B with habitual FIX coagulant activities of 10-12% and FIX antigen levels of 50%. We found that activation by FVIIa/TF and FXIa was normal for FIXPro55Ser, but resulted in proteolysis of FIXPro55Leu at Arg318-Ser319 with a concomitant loss of amidolytic activity, suggesting intramolecular communication between EGF1 and the serine protease domain in FIX. This was further supported by experiments using an anti-EGF1 monoclonal antibody. Activation of FX by FIXaPro55Ser was impaired in both the presence and the absence of phospholipid or FVIIIa, indicating that Pro55 is not directly involved in binding to FVIIIa. We also studied the effect of the two Pro55 mutations on Ca2+ affinity and found only small changes. Thus, the Pro55Ser mutation causes hemophilia primarily through to an impaired ability to activate FX whereas at least in vitro the Pro55Leu defect interferes with the activation of FIX.

Calcium↗
PubMed2003

A modified thrombin generation test for the measurement of factor VIII concentrates.

It has been well documented that there is an uncertainty over the true factor (F)VIII level in postinfusion samples due to assay discrepancies. The thrombin generation test (TGT) was used as a potentially more physiological approach to assess and compare FVIII concentrates. FVIII concentrates were added to artificial FVIII-deficient plasma. Thrombin generation was initiated by the addition of FIXa (14 nm), phospholipid and CaCl2. Thrombin was measured by subsampling into fibrinogen, and curves quantified as area under the curve (AUC) and time taken to half-maximum (t(1/2)max). Addition of one plasma-derived concentrate to as little as 0.005 IU mL-1 gave a normal AUC, but prolonged t(1/2)max. Increasing FVIII to 1 IU mL-1 had little effect on AUC, but did reduce the t(1/2)max to 64 s (normal 114 s). A range of plasma-derived and recombinant concentrates were tested at 1 IU mL-1; results were similar, except the B-domain deleted concentrate, which had the most rapid initial rate of thrombin generation (t(1/2)max 48 s, P < 0.05). Two hemophilic plasmas (< 0.01 IU mL-1) produced large amounts of thrombin (AUC 65% and 69%), although t(1/2)max was prolonged. Addition of a FVIII antibody abolished thrombin generation, indicating that these plasmas contained low levels of FVIII. Decreasing the FIXa concentration (0.2 nm) minimized thrombin generation in hemophilic plasma but not in normal plasma. These results indicate that FVIII < 0.01 IU mL-1 can generate significant quantities of thrombin depending upon the amount of FIXa present. The TGT could prove useful for patient monitoring in gene therapy and prophylaxis.

Blood Coagulation Tests↗
PubMed2003

What is all that thrombin for?

The hemostatic process initiated by the exposure of tissue factor to blood is a threshold limited reaction which occurs in two distinct phases. During an initiation phase, small amounts of factor (F)Xa, FIXa and thrombin are generated. The latter activates the procofactors FV and FVIII to the activated cofactors which together with their companion serine proteases form the intrinsic FX activator (FVIIIa-FIXa) and prothrombinase (FVa-FXa) which generate the bulk of FXa and thrombin during a propagation phase. The clotting process (fibrin formation) occurs at the inception of the propagation phase when only 5-10 nM thrombin has been produced. Consequently, the vast majority (greater than 95%) of thrombin is produced after clotting during the propagation phase of thrombin generation. The blood of individuals with either hemophilia A or hemophilia B has no ability to generate the intrinsic FXase, and hence is unable to support the propagation phase of the reaction. Since clot based assays conclude before the propagation phase they are not sensitive to hemophilia A and B. The inception and magnitude of the propagation phase of thrombin generation is influenced by genetic polymorphisms associated with thrombotic and hemorrhagic disease, by the natural abundance of pro- and anticoagulants in healthy individuals and by pharmacologic interventions which influence thrombotic pathology. Therefore, it is our suspicion that the performance of the entire process of thrombin generation from initiation through propagation and termination phases of the reaction are relevant with respect to both hemorrhagic and thrombotic pathology.

Animals↗
PubMed2002

Overview of anticoagulant drugs for the future.

More efficacious, safer, and easier to use anticoagulants are under development. Multiple agents have been shown to be effective in ex vivo or animal thrombosis models and several have progressed to clinical studies. Investigators have not yet determined if pharmaceuticals that inhibit coagulation factor activity earlier in the cascade (for example, inhibitors of tissue factor/factor VIIa, factor IXa, or Xa) are superior to those that block the cascade at a later point. Orally bioavailable drugs for the long-term treatment of thrombotic disorders, particularly those that do not require monitoring, are needed and are under development. Local delivery of anticoagulants or genes modulating anticoagulant control at sites of increased thrombogenicity, such as in diseased arteries, is a promising treatment modality that may decrease systemic bleeding problems. Much about the initiating pathophysiologic events leading to venous thrombotic disease needs to be elucidated before such local therapy can be tested in the venous vasculature. While awaiting better anticoagulants to become routinely available, we need to improve patient management with existing drugs by instituting anticoagulation clinics, promoting patient self-monitoring, and improving efforts to educate patients and health care providers about the use of anticoagulant drugs.

Anticoagulants↗
PubMed1986

Modulation of smooth muscle responses by serine proteases and related enzymes.

Standard isolated smooth muscle tissue preparations were used to screen the musculotropic actions of serine proteases and other substances that are generated during hemostatic activation. Sera obtained from human, rabbit, and guinea pigs produced a dose-dependent contraction of these isolated tissue preparations. These sera also augmented the actions of epinephrine and other agonists on different tissue preparations. Both contractile and relaxant actions of proteases were observed with various serine proteases and their complexes. Thrombins (alpha, beta, and gamma) produced varying degrees of contractile actions of rabbit aortic strips; however, Factor Xa produced a marked relaxation of this preparation. Trypsin and kallikrein did not produce any action on the aortic strip and thrombin, however, produced contraction of the guinea pig ileum. Protease complexes produced varying musculotropic actions on the isolated tissue preparations, which may be related to their composition. The musculotropic actions of serum and thrombin were not blocked by conventional pharmacologic antagonists. However, protease inhibitors, such as hirudin, D-Phe-Pro-Arginal, and other derivatives, produced varying effects of the musculotropic actions of serum and thrombins. These results indicate that thrombin and related proteases generated during the activation of the hemostatic system are capable of producing direct musculotropic actions of various smooth muscles. Various serine proteases and serine protease complexes were found to produce strong hemodynamic effects in a rabbit model for the study of blood plasma. The smooth muscle modulant actions of serine proteases should be taken into account to clarify the vascular pathologic changes in relation to spasmogenic and spasmolytic syndromes observed during hemostasis disorders.

Animals↗
PubMed1981

The effect of activated prothrombin-complex concentrate (FEIBA) on joint and muscle bleeding in patients with hemophilia A and antibodies to factor VIII. A double-blind clinical trial.

We designed a double-blind trial to study the effect of an "activated" prothrombin-complex concentrate (FEIBA) on joint and muscle bleeding in hemophiliacs with antibodies to factor VIII. Fifteen patients received either FEIBA or the control preparation (a nonactivated prothrombin-complex concentrate) for a total of 150 bleeding episodes (four mucocutaneous bleeding, 117 joint bleeding, and 29 muscle bleeding). In 64 per cent of the episodes, FEIBA was judged by the physician to be effective; the control preparation was perceived as effective in 52 per cent of the episodes in which it was used. Pairwise comparison of FEIBA and the control preparation for bleeding in the same joint or muscle showed a significantly better result with FEIBA (P = 0.0085). Joint mobility after the use of FEIBA was significantly improved (P = 0.006). There was a high incidence of hepatitis (three of the 15 patients) and of transient disturbances of liver function (nine of 15) during the 15-month observation period.

Adolescent↗
PubMed1990

Activation of coagulation after administration of tumor necrosis factor to normal subjects.

Tumor necrosis factor has been implicated in the activation of blood coagulation in septicemia, a condition commonly associated with intravascular coagulation and disturbances of hemostasis. To evaluate the early dynamics and the route of the in vivo coagulative response to tumor necrosis factor, we performed a controlled study in six healthy men, monitoring the activation of the common and intrinsic pathways of coagulation with highly sensitive and specific radioimmunoassays. Recombinant human tumor necrosis factor, administered as an intravenous bolus injection (50 micrograms per square meter of body-surface area), induced an early and short-lived rise in circulating levels of the activation peptide of factor X, reaching maximal values after 30 to 45 minutes (mean +/- SEM increase after 45 minutes, 34.2 +/- 18.2 percent; tumor necrosis factor vs. saline, P = 0.015). This was followed by a gradual and prolonged increase in the plasma concentration of the prothrombin fragment F1+2, peaking after four to five hours (mean increase after five hours, 348.0 +/- 144.8 percent; tumor necrosis factor vs. saline, P less than 0.0001). These findings signify the formation of factor Xa (activated factor X) and the activation of prothrombin. Activation of the intrinsic pathway could not be detected by a series of measurements of the plasma levels of factor XII, prekallikrein, factor XIIa-C1 inhibitor complexes, kallikrein-C1 inhibitor complexes, and the activation peptide of factor IX. The delay between the maximal activation of factor X and that of prothrombin amounted to several hours, indicating that neutralization of factor Xa activity was slow. We conclude that a single injection of tumor necrosis factor elicits a rapid and sustained activation of the common pathway of coagulation, probably induced through the extrinsic route. Our results suggest that tumor necrosis factor could play an important part in the early activation of the hemostatic mechanism in septicemia.

Adult↗
PubMed1997

Endothelial cell thrombin receptors and PAR-2. Two protease-activated receptors located in a single cellular environment.

Human endothelial cells express thrombin receptors and PAR-2, the two known members of the family of protease-activated G protein-coupled receptors. Because previous studies have shown that the biology of the human thrombin receptor varies according to the cell in which it is expressed, we have taken advantage of the presence of both receptors in endothelial cells to examine the enabling and disabling interactions with candidate proteases likely to be encountered in and around the vascular space to compare the responses elicited by the two receptors when they are present in the same cell and to compare the mechanisms of thrombin receptor and PAR-2 clearance and replacement in a common cellular environment. Of the proteases that were tested, only trypsin activated both receptors. Cathepsin G, which disables thrombin receptors, had no effect on PAR-2, while urokinase, kallikrein, and coagulation factors IXa, Xa, XIa, and XIIa neither substantially activated nor noticeably disabled either receptor. Like thrombin receptors, activation of PAR-2 caused pertussis toxin-sensitive phospholipase C activation as well as activation of phospholipase A2, leading to the release of PGI2. Concurrent activation of both receptors caused a greater response than activation of either alone. It also abolished a subsequent response to the PAR-2 agonist peptide, SLIGRL, while only partially inhibiting the response to the agonist peptide, SFLLRN, which activates both receptors. After proteolytic or nonproteolytic activation, PAR-2, like thrombin receptors, was cleared from the endothelial cell surface and then rapidly replaced with new receptors by a process that does not require protein synthesis. Selective activation of either receptor had no effect on the clearance of the other. These results suggest that the expression of both thrombin receptors and PAR-2 on endothelial cells serves more to extend the range of proteases to which the cells can respond than it does to extend the range of potential responses. The results also show that proteases that can disable these receptors can distinguish between them, just as do most of the proteases that activate them. Finally, the residual response to SFLLRN after activation of thrombin receptors and PAR-2 raises the possibility that a third, as yet unidentified member of this family is expressed on endothelial cells, one that is activated by neither thrombin nor trypsin.

Endopeptidases↗
PubMed1999

Identification of amino acids in the factor XI apple 3 domain required for activation of factor IX.

Activated coagulation factor XI (factor XIa) proteolytically cleaves its substrate, factor IX, in an interaction requiring the factor XI A3 domain (Sun, Y., and Gailani, D. (1996) J. Biol. Chem. 271, 29023-29028). To identify key amino acids involved in factor IX activation, recombinant factor XIa proteins containing alanine substitutions for wild-type sequence were expressed in 293 fibroblasts and tested in a plasma clotting assay. Substitutions for Ile(183)-Val(191) and Ser(195)-Ile(197) at the N terminus and for Ser(258)-Ser(264) at the C terminus of the A3 domain markedly decreased factor XI coagulant activity. The plasma protease prekallikrein is structurally homologous to factor XI, but activated factor IX poorly. A chimeric factor XIa molecule with the A3 domain replaced with A3 from prekallikrein (FXI/PKA3) activated factor IX with a K(m) 35-fold greater than that of wild-type factor XI. FXI/PKA3 was used as a template for a series of proteins in which prekallikrein A3 sequence was replaced with factor XI sequence to restore factor IX activation. Clotting and kinetics studies using these chimeras confirmed the results obtained with alanine mutants. Amino acids between Ile(183) and Val(191) are necessary for proper factor IX activation, but additional sequence between Ser(195) and Ile(197) or between Phe(260) and Ser(265) is required for complete restoration of activation.

Amino Acid Sequence↗
PubMed2002

Role of the N-terminal EGF module of coagulation factor IX in activation of factors IX and X.

Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder haemophilia B. FIX contains a Gla module, two epidermal growth factor-like (EGF) modules, and a serine protease region. I characterized a monoclonal antibody and found that it recognizes an epitope around residues 72 and 80 in the C-terminal part of EGF1 in human FIX. The antibody exhibited 10-fold greater affinity for activated FIX (FIXa) than for the zymogen FIX, indicating the existence of intra-molecular communication between the serine protease region and EGF1. Binding of the antibody did not affect the amidolytic activity of FIXa, hence I could use the antibody during activation of FIX to show that the C-terminal part of EGF1 is of importance for the interaction with FXIa but not with FVIIa/TF. Considering activation of FX, it is a matter of debate whether EGF1 or FIXa interacts directly with FVIIIa. I activated FX in the presence and absence of the antibody and/or FVIIIa. The addition of antibody caused only a minor decrease in k(cat,app), and the major increase in k(cat,app) caused by the addition of FVIIIa occurred even in the presence of the antibody. This implies that EGF1 of FIXa is not directly involved in interaction with FVIIIa in the Xase complex. A model of the FIXa-FVIIIa complex, based on my findings and results from the literature, was constructed and indicated that EGF1 of FIXa does not interact directly with FVIIIa.

Amino Acid Sequence↗