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PubMed1990

Molecular defect (Gla+14----Lys) and its functional consequences in a hereditary factor X deficiency (factor X "Vorarlberg").

Factor X (FX) "Vorarlberg" is a congenital FX deficiency characterized clinically by a mild bleeding tendency. Homozygous individuals have a FX activity of less than 10% in the extrinsic system and 25% in the intrinsic system. FX antigen is 20%. Using molecular techniques, two point mutations were detected in the coding sequence of the FX Vorarlberg gene: a G----A at base pair 160 in exon II resulting in a change of Gla14 (GAA) to Lys (AAA); a G----A at base pair 424 in exon V resulting in a change from Glu102 (GAG) to Lys (AAG). The mutations abolished a TaqI restriction site in exon II and an MnlI site in exon V. To determine whether these mutations are present on one or on both alleles, restriction analyses of amplified exon II and exon V fragments were performed. Analysis of the pedigree showed that the genotype for the mutation on exon II (homozygous versus heterozygous) correlates with the severity of the phenotypic coagulation defect. We therefore conclude that the mutation in exon II is responsible for the functional defect in FX Vorarlberg. We have also purified the mutant FX protein from patient plasma. Purified FX Vorarlberg is indistinguishable from normal FX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity is 15% of normal FX upon activation with factor VIIa/tissue factor, 75% upon activation with factor IXa/factor VIIIa, and 100% upon activation with RVV. Activation at varying Ca2+ concentrations shows that the affinity of FX Vorarlberg for Ca2+ is decreased. Factor Xa Vorarlberg is able to convert prothrombin at a normal rate but also shows decreased affinity for Ca2+ in this interaction. Upon addition of Ca2+, FX Vorarlberg does not undergo the same conformational change as normal FX. Our data show that FX Vorarlberg has a decreased affinity for Ca2+ which impedes a normal conformational change. This leads to a decreased rate of activation by factor VIIa/tissue factor and by factor IXa. The decrease is much more marked for the extrinsic than for the intrinsic pathway.

Amino Acid Sequence↗
PubMed1987

A simple and accurate microplate assay for the determination of factor VIII activity.

Based on the CoatestR Factor VIII kit, a simple and accurate microtiter plate assay has been developed. The method has been simplified through the combination of the bovine factors IXa + X, phospholipid and calcium chloride into one reagent. A further improvement was obtained by generating F Xa to a stable plateau level, thus minimizing influences from variations in time and temperature. The substrate hydrolysis was terminated with 1 M citrate buffer, pH 3.0, which reduced the environmental effects as compared to acetic acid. Overall a fourfold reduction in reagent consumption was achieved. A high correlation with clotting assays was obtained with various types of factor VIII concentrates as well as with plasma samples from blood donors and hemophilia A patients (r greater than 0.85). The accuracy was proven over the whole investigated range of factor VIII activities. Finally, the described microplate assay allows a high turnover of samples in a short period of time, still maintaining a coefficient of variation below 5%.

Blood Coagulation Tests↗
PubMed1994

Quality of plasma separated from (buffy coat + plasma). An alternative way of blood processing.

A CPD/ADSOL triple-bag system was used to produce plasma, buffy coat and resuspended erythrocytes. These components could be produced in a quadruple-bag system when working according to the conventional technique. In the experimental technique, buffy coat and plasma are transferred together into the satellite bag and are separated from each other only after the second centrifugation. The plasma complement system is not activated and factor IXa is not generated when applying the experimental technique. The quality of plasma meets the international requirements. The blood component processing technique using a triple-bag system is less expensive compared to the quadruple-bag one.

Blood Coagulation Factors↗
PubMed1992

Dibucaine elicits platelet procoagulant activity in factor VIII and factor X activation by a mechanism involving a sulfhydryl-dependent enzyme.

Dibucaine, a potent inhibitor of platelet aggregation and platelet release, was found to enhance the ability of fresh gel-filtered or washed human platelets to support factor VIII activation and factor X activation. Dibucaine-treated platelets increased the peak of factor VIII clotting activity by 2-fold compared to activity with untreated platelets. Similarly platelets optimally stimulated by dibucaine (1.0-1.5 mM for 5 min at 37 degrees C) supported as much factor X activation by factors IXa and VIII (measured in a chromogenic assay) as platelets optimally stimulated by ionophore A23187 (15 microM). An assay of platelet calcium-dependent sulfhydryl proteases was devised and used to test the effect of various inhibitors on these platelet proteases. The membrane-permeable sulfhydryl inhibitor Thiolyte MB inhibited platelet calcium-dependent protease activity; whereas, membrane-impermeable Thiolyte MQ did not. Thiolyte MB also blocked the ability of dibucaine-stimulated platelets to support factor X activation. Incubation of fresh, gel-filtered platelets with calpain inhibitor II (N-Ac-L-L-Normethioninal) completely inhibited the calcium-dependent sulfhydryl protease activity of these platelets but did not affect their ability to support factor X activation after subsequent incubation with dibucaine. These data support the interpretation that an intracellular SH-dependent enzyme, which may not be calpain, is involved in the expression of platelet procoagulant activity in dibucaine-treated platelets.

Bridged Bicyclo Compounds↗
PubMed2001

In hemophilia A and autoantibody inhibitor patients: the factor VIII A2 domain and light chain are most immunogenic.

Factor VIII (fVIII) is a protein cofactor essential for blood coagulation, and it binds in the factor Xase complex to factors IXa, X, and phospholipid. In about 30% of severe hemophilia A patients, treatment with fVIII leads to production of anti-fVIII antibodies. Anti-fVIII autoantibodies also rarely appear in normal individuals. Those antibodies that inactivate fVIII (inhibitors) prevent optimal fVIII therapy. Inhibitor epitopes were previously localized to the fVIII A2, A3, and C2 domains and to an acidic amino acid region between A1 and A2. Such anti-fVIII antibodies interfere with fVIII binding to components of the factor Xase complex and prevent blood coagulation. When total anti-fVIII titers were determined for each fVIII domain in 43 inhibitor plasmas by immunoprecipitation (IP) and inhibitor neutralization assays, the anti-light chain (LCh) antibody titer was highest, anti-A2 was intermediate, and anti-A1 and anti-B were low. The relative immunogenicity of the fVIII domains in hemophilic and autoantibody inhibitor patients was similar.

Antibodies, Monoclonal↗
PubMed1979

[Pharmacology of heparin].

After introductory notes on the history of heparin research the chemistry of this mucopolysaccharide is described. It is shown that the chemistry of heparin cannot be exactly described by one chemical formula because heparin represents a family of compounds with different chain-length. Molecular features responsible for the high structural specificity of anticoagulant activity are described. The present status of knowledge on the mechanism of the anticoagulant action of heparin is described in detail. It is shown that basic mechanism is the acceleration of the interaction of antithrombin III with the clotting factors IXa, Xa, XIa, XIIa, and thrombin which are known to be serine proteases. The effects of heparin of platelet function, lipid composition of blood, and on fibrinolysis reflect its complex influence on hemostasis. After a description of side effects and toxicity clinically relevant data on its pharmacocinetics are given.

Animals↗
PubMed2004

Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases.

A unique pentasaccharide fragment of heparin can enhance the reactivity of antithrombin with coagulation proteases factors IXa and Xa by 300- to 600-fold through a conformational activation of the serpin, without having a significant effect on the reactivity of antithrombin with thrombin. In this study, it was hypothesized that differences in the structure of the autolysis loop of coagulation proteases (residues 143-154 in chymotrypsin numbering) may be responsible for their differential reactivity with the native and heparin-activated antithrombin. To test this hypothesis, the autolysis loops of both thrombin and the anticoagulant serine protease-activated protein C were replaced with the corresponding loop of factor Xa. Inhibition studies revealed that in contrast to the approximately 1.5-fold difference in the reactivity of thrombin with antithrombin in the absence and presence of pentasaccharide, the difference in reactivity was increased to approximately 37-fold for the mutant thrombin. In the case of the activated protein C mutant, similar to factor Xa, pentasaccharide accelerated the reaction 375-fold. These results suggest that structural differences in the autolysis loop of coagulation proteases play a key role in their differential reactivity with the native and heparin-activated conformations of antithrombin.

Animals↗
PubMed1995

In vivo models of thrombogenic potential: usefulness and limitations.

The thrombogenicity of prothrombin complex concentrates (PCCs) has been known as a risk factor since their first clinical use about 30 years ago. The development of in vivo models to define the thrombogenic components in PCCs was instrumental in providing a logical basis for selecting in vitro assays to screen for the distribution of such components during the manufacture of PCCs, and to minimize their appearance in the final product. Even so, these thrombogenic components are not completely removed, as shown in our canine nonstasis model of thrombogenicity: PCCs were still found to elicit a thrombogenic response, shown by increased fibrinopeptide A, fibrin(ogen) degradation products, activated partial thromboplastin time, and decreased fibrinogen and platelet counts when clinically relevant doses were used. The new generation of high-purity factor IX (HP-FIX) concentrates differs from PCCs because these products contain only negligible amounts of clotting factors other than factor IX, lower amounts of activated clotting factors, and, in products we have assayed, no coagulant-active phospholipids. When we infused a number of HP-FIX products in the canine nonstasis model, no thrombogenic response was observed at doses considerably greater than PCC doses that did elicit a response. Likewise, HP-FIX products were much less thrombogenic than PCCs when tested in small-animal stasis and nonstasis thrombogenicity models. Small-animal models are also useful for evaluating the role of factor IXa as a potential thrombogenic contaminant of concentrates and ensuring minimal amounts in the final product. The limitations associated with extrapolating in vivo model data will be shown to be minimal if ongoing clinical studies continue to demonstrate the low thrombogenic potential of HP-FIX concentrates in humans.

Animals↗
PubMed2002

The interaction of factor XIa with activated platelets but not endothelial cells promotes the activation of factor IX in the consolidation phase of blood coagulation.

We have previously shown that the zymogen factor XI (FXI) binds to activated platelets but not to human umbilical vein endothelial cells (HUVEC), a conclusion that is in conflict with previous reports stating that FXI binds to 2.7-13 x 10(6) high affinity sites per HUVEC (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839; Shariat-Madar, Z., Mahdi, F., and Schmaier, A. H. (2001) Thromb. Haemostasis 85, 544-551). It has also been reported that activated FXI (FXIa) binds to 1.5 x 10(6) sites per HUVEC and promotes the activation of factor IX by cell bound FXIa (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839). Therefore, the binding of FXIa to activated platelets was compared with FXIa binding to HUVEC and HEK293 cells immobilized on microcarrier beads. Specific and saturable zinc-dependent FXIa binding was demonstrated to 250 +/- 48 sites per activated platelet (K(D) = 1.7 +/- 0.78 nm) and 6.5 +/- 0.4 x 10(4) sites per HUVEC (K(D) = 2.4 +/- 0.5 nm), whereas no binding to HEK293 cells was detected. A titration with high molecular weight kininogen had no effect on FXIa binding to platelets, but revealed a concentration-dependent decrease in the amount of FXIa bound to HUVEC. The rate of factor IXa generation catalyzed by FXIa was unaffected by the presence of surfaces; however only the activated platelet surface protected FXIa from inhibition by protease nexin 2. The results presented here confirm the conclusion that activated platelets are procoagulant while unstimulated endothelial cells are not.

Amyloid beta-Protein Precursor↗
PubMed1979

Minimal intermittent heparinization during hemodialysis.

Minimal dose heparinization inhibiting clotting factor IXa, Xa, XIa, as monitored by the activated partial thromboplastin time, was compared with conventional intermittent, continuous and regional heparinization during hemodialysis treatment. Blood loss in coil dialyzers was the same. Heparin dosage was reduced markedly. Protamine sulfate and infusion equipment were not required. No bleeding problems were encountered in high-risk patients.

Dose-Response Relationship, Drug↗
PubMed2001

Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity.

Factor VII requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active factor VIIa (FVIIa) conformation. The former event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. We have designed a number of FVIIa mutants with the aim of mimicking the effect of TF, that is, creating molecules with increased intrinsic (TF-independent) enzymatic activity. Based on a possible structural difference between free and TF-bound FVIIa (Pike, A. C. W., Brzozowski, A. M., Roberts, S. M., Olsen, O. H., and Persson, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8925--8930), we focused on the helical region encompassing residues 307-312 and residues in its spatial vicinity. For instance, FVIIa contains Phe-374 and Leu-305, whereas a Phe/Tyr residue in the position corresponding to 374 in homologous coagulation serine proteases is accompanied by Val in the position corresponding to 305. This conceivably results in a unique orientation of this helix in FVIIa. Substitution of Val for Leu-305 in FVIIa resulted in a 3--4-fold increase in the intrinsic amidolytic and proteolytic activity as compared with wild-type FVIIa, whereas the activity in complex with soluble TF remained the same. In accordance with this, L305V-FVIIa exhibited an increased rate of inhibition as compared with wild-type FVIIa, both by d-Phe-Phe-Arg-chloromethyl ketone and antithrombin III in the presence of heparin. The increased FVIIa activity upon replacement of Leu-305 by Val may be mediated by a movement of the 307--312 helix into an orientation resembling that found in factors IXa and Xa and thrombin. The corresponding shortening of the side chain of residue 374 (Phe --> Pro) had a smaller effect (about 1.5-fold increase) on the intrinsic activity of FVIIa. Attempts to increase FVIIa activity by introducing single or multiple mutations at positions 306, 309, and 312 to stabilize the 307-312 helix failed.

Amino Acid Substitution↗
PubMed1986

Specificity of the thrombin-induced release of tissue plasminogen activator from cultured human endothelial cells.

The addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of alpha-thrombin, promoted tPA release but was less effective than alpha-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of gamma-thrombin 20 times greater than alpha-thrombin was required. The response to Factor Xa was similar to alpha-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active alpha-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

Cells, Cultured↗
PubMed1988

Partial deletion by illegitimate recombination of the factor IX gene in a haemophilia B family with two inhibitor patients.

The inhibitor phenotype occurs in six haemophilia B patients in the UK and results from development of antibodies by the patients to administered factor IX. We have analysed a partial factor IX gene deletion (London 1) in a family with two inhibitor patients. The deletion results in retention of the first five exons which code for the light chain of factor IXa, and removal of 23 kb of DNA starting 704 bp 3' of the fifth exon and terminating 10.3 kb 3' of the last exon. The 5' break is at residue -113 of an Alu repeat. No significant homology exists between the 5' and 3' termini, but a 9 bp region of complementarity is found 23 bp and 60 bp from the 5' and 3' terminus, respectively. At the cloned deletion junction a new 16 bp sequence contributes a DraI site that is also found in the genomic DNA of the two patients and a heterozygous relative. The deletion is an example of illegitimate recombination and it is proposed that such deletions occur principally during DNA replication. Loss of the 3' sequences involved in the maturation of mRNA probably results in no factor IX production. Immunological studies show that the index patient's antibodies bind both to epitopes coded by deleted and by non-deleted segments of the gene.

Base Sequence↗
PubMed1999

Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function.

The functional defect caused by substitution of Arg527 (--> Trp) and Arg531 (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527.

Amino Acid Substitution↗
PubMed1984

Location and partial characterization of the heparin-binding fragment of platelet thrombospondin.

Purified platelet thrombospondin (TS) was subjected to proteolysis with a number of proteases including factors IXa, Xa, thrombin, elastase, trypsin, and chymotrypsin. All enzymes yielded fragments of TS which bound to heparin-Sepharose. Only chymotrypsin cleavage produced a single species of heparin-binding fragment, as analyzed by SDS-PAGE. This fragment had a chain molecular weight of 28,000, and contained no interchain disulfide bonds. Amino acid sequence analysis of the heparin-binding fragment and of TS revealed a single sequence, indicating that the fragment constitutes the amino-terminal domain of TS and that the three chains in TS are identical in this region.

Amino Acid Sequence↗
PubMed2006

Emerging anticoagulants: mechanism of action and future potential.

Medical needs associated with diverse thromboembolic conditions are not fully met by currently available anticoagulants. Of those, unfractionated heparin (UFH) is gradually replaced by low molecular weight heparin (LMWH) for prevention and treatment of venous thromboembolism and acute coronary syndromes, along with supportive treatment with oral anticoagulants, such as warfarin derivatives. While generally effective these agents have several shortcomings involving compliance, delivery, efficacy and safety considerations in various disease settings, and for these reasons new anticoagulants are sought, to target more specifically the critical effectors and steps in the blood coagulation process, namely: (i) initiation, (ii) propagation and (iii) the phase of thrombin activity. The emerging agents that block tissue factor/factor VIIa-dependent initiation phase of the coagulation cascade, include: recombinant tissue factor pathway inhibitor (rTFPI), nematode anticoagulant peptide (NAPc2), active site-blocked factor VIIa (FVIIai) and TF targeting antibodies. Some of them are currently evaluated in clinical trials with promising results. Propagation phase of thrombus formation (e.g. the activity of factors IXa, Xa, VIIIa or Va) is targeted mainly by various indirect, direct and bimodal inhibitors, such as fondaparinux, indraparinux, tick anticoagulant peptide (TAP), antistatin (ANT) and antithrombin-heparin covalent complex (ATH), all endowed mostly with an anti-Xa activity. Although promising, some of these agents (TAP, ANT and ATH) have not progressed beyond animal testing while others (fondaparinux) was already assessed for prevention and treatment of venous thromboembolism and for treatment of arterial thrombosis. Lastly, inhibitors of thrombin activity are composed of either indirect (UFH, LMWH), or direct thrombin (FIIa) inhibitors including: hirudin, argatroban, melagatran, ximelagatran, dabigatran, and bivalirudin. These agents are either in advanced development or already approved for clinical use. Bimodal FIIa inhibitory activity of ATH was demonstrated in animal models of venous and arterial thrombosis, but is in need of further development. In conclusion, while some of these emerging anticoagulants, such as fondaparinux, idraparinux, ximelagatran and ATH appear to possess superior efficacy-safety profile, as compared to their conventional predecessors (UFH, LMWH and warfarin), their cost-effectiveness, side effects and antidote availability have to be considered. More importantly, coagulation factors that are targets of these inhibitory activities also affect coagulation independent processes, such as wound healing, inflammation, angiogenesis, mitogenesis and cell survival. Thus the consequences of both coagulation-dependent and -independent effects of new agents should be carefully considered before proper clinical indications are established.

Anticoagulants↗
PubMed2002

Anticoagulant effects of a synthetic peptide containing residues Thr-2253-Gln-2270 within factor VIII C2 domain that selectively inhibits factor Xa-catalysed factor VIII activation.

Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti-FVIII C2 antibody (ESH8) with a Val-2248-Gly-2285 epitope inhibited FXa-catalysed FVIII activation, and that a synthetic peptide designated EP-2 (residues 2253-2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP-2 on FXa-catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP-2 inhibited FXa-catalysed activation in a clotting assay in a dose-dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP-2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose-dependent manner, but not prothrombin time. Our results indicate that EP-2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa-catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy.

Anticoagulants↗
PubMed1990

Two mutations of the factor IX gene including a donor splice consensus deletion and a point mutation in a Dutch patient with severe hemophilia B.

The abnormal factor IX gene of a patient with severe hemophilia B (hemophilia B Ursem) was selected for study. All of the coding and their flanking regions and parts of the 5'- and 3'-untranslated regions of the factor IX gene were amplified from the patient's genomic DNA by using the polymerase chain reaction (PCR). By analyzing the nucleotide sequence of the PCR products we have identified two mutations in the patient's factor IX gene, viz. a tetranucleotide deletion (GAGT, nt 6492 to 6495) or (TGAG, nt 6491 to 6494) in the 5'-donor splice site consensus at the exon 2-intron B boundary, and a point mutation at nucleotide 31103 in the catalytic domain (exon 8) of factor IXa, which changes the codon for valine 328 (GTT) to one for isoleucine (ATT). PCR-amplified exon 8 from 45 normal males and 55 normal females had the codon for valine-328. We propose that the deletion within the donor splice-site consensus is the cause of the disease in this individual, whereas the substitution of valine-328 by isoleucine may be a neutral variant which is, at least, very rare in the normal population. In a family study the DNA sequence of the patient's mother shows both the G to A transition in exon 8 and the 5'-donor splice consensus deletion in intron B in one allele.

Amino Acid Sequence↗