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PubMed2006

Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor of reaction mechanism.

Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor.enzyme template bridging by heparin. Rationales behind this novel concept are discussed.

Antithrombins↗
PubMed1994

Protein engineering of the hydrophobic domain of human factor IX.

Vitamin K-dependent plasma proteins contain a highly conserved hydrophobic domain located between the gamma-carboxyglutamic acid (Gla) domain and the first epidermal growth factor (EGF)-like domain. Here we have used protein engineering of the hydrophobic domain in human factor IX to investigate its function in intact factor IX. Mutant proteins were generated by site-directed mutagenesis and in vitro expression in Madin-Darby canine kidney (MDCK) cells. All of our mutants, including one with a deletion of the entire hydrophobic domain, were activated by factor XIa, showing that this domain is not required for factor IX activation. The results with the mutant Phe41-->Val suggest that the hydrophobic domain interacts with the adjacent EGF-like domain. Our data for the Phe41-->Asp mutant is consistent with, but cannot prove, a role for this residue in the maintenance of a phospholipid-binding structure required for factor IX function.

Amino Acid Sequence↗
PubMed2002

Activation of factor IX by erythrocyte membranes causes intrinsic coagulation.

As an extension of our earlier work, procoagulant activity of erythrocyte [red blood cell (RBC)] membrane was examined using biochemical and rheological techniques. Western blot analysis of coagulation factors incubated with erythrocytes (RBCs) showed that only factor IX (FIX) was activated by RBC membranes in the presence of calcium ions. A fluorogenic assay suggested that activated FIX is capable of activating factor X. A preliminary crude extraction of the substance from RBC membranes suggested that a FIX-activating enzyme may be located on the RBC membrane. The initiation of FIX activation by RBCs was enhanced by an elevation in hematocrit. Moreover, the rate of FIX activation by RBCs from normal pregnant women and diabetic patients was much faster than that from normal subjects. In addition, glucose treatment of normal RBCs resulted in the increase in procoagulant activity. It is suggested that FIX activation by RBC membranes may serve as a triggering mechanism for blood coagulation, although further study will be required to clarify the putative FIX activating enzyme on the RBC membrane and to permit more extensive physiological experiments to be performed.

Blood Coagulation↗
PubMed1983

The interaction between factor VIII clotting antigen (VIIICAg) and phospholipid.

The interaction between factor VIII clotting antigen (VIIICAg) and phospholipid (PL) was studied using a two-site solid phase immunoradiometric assay (IRMA) for VIII CAg. Incubation (2 h, 37 degrees C) of normal plasma, cryoprecipitate or factor VIII concentrate with Diagen PL (0.5 mg/unit VIIICAg) resulted in 80-90% loss of IRMA-measurable VIIICAg. No loss of factor VIII related antigen (VIIIRAg) or factor VIII clotting activity (VIIIC) was seen. Treatment of factor VIII concentrate with purified PLs showed greatest VIIICAg reduction with phosphatidylserine, less with phosphatidylethanolamine, and very little with phosphatidylcholine. The action of phospholipase-C (PL-C) on VIIICAg-PL complexes was investigated, with enzyme activity being monitored by thin-layer chromatography. Treatment of normal plasma, cryoprecipitate or factor VIII concentrate with PL-C (5 u/unit VIIICAg) resulted in 25%, 25% and 30% increases in VIIICAg. No increase in VIIIC or VIIIRAg was seen. Preincubation of factor VIII concentrate with PL, followed by PL-C incubation, resulted in 70-80% recovery of measurable VIIICAg. Incubation of 'activated' prothrombin complex with PL-C increased VIIICAg by 44% (Autoplex) and 80% (FEIBA), indicating VIIICAg-PL complexes are present. Incubation of factor VIII concentrate with fresh platelet lysate led to a reduction in VIIICAg (100 u/dl to 21 u/dl). In fresh platelet lysate alone low VIIICAg levels were detectable (0.71 X 10(-3) u/10(9) plt). After PL-C incubation VIIICAg levels increased to 9.8 X 10(-3) u/10(9) plt) (14-fold increase). Thus VIIICAg in platelets may be hidden by VIIICAg-PL complexes.

Antigens↗
PubMed1993

Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.

Recombinant factor VIII variants with overlapping deletions spanning the region Lys713-Ile1668 have been expressed in mammalian cells, and analysed for biological activity both in vitro and in vivo. Two distinct assay systems were used to measure the activity in vitro. The one-stage coagulation assay served to assess factor VIII procoagulant activity while a spectrophotometric assay was used for the quantification of factor VIII cofactor activity in factor IXa-dependent factor X activation. Deletion of the entire B-domain (Ser741-Arg1648) resulted in a protein with similar procoagulant and cofactor activity. In contrast, factor VIII-del(713-1637), which has a deletion that also comprises the heavy-chain sequence Lys713-Arg740, had lost factor VIII procoagulant activity while factor VIII cofactor activity was retained. This functional inconsistency was further addressed by comparing purified factor VIII-del(713-1637) with factor VIII-del(868-1562), a mutant with normal in vitro activity. Kinetic studies of factor Xa formation revealed that higher concentrations of thrombin were required to develop the cofactor activity from factor VIII-del(713-1637) than needed for factor VIII-del(868-1562) or plasma factor VIII. The physiological significance of this finding was assessed in dogs with haemophilia A. Both deletion mutants were similar to plasma factor VIII with regard to binding to von Willebrand factor and half-life and recovery. Employing the cuticle bleeding time model, factor VIII-del(868-1562) was found to be indistinguishable from plasma factor VIII, whereas factor VIII-del(713-1637) was less effective. The increased thrombin-resistance of factor VIII-del(713-1637) thus limits both procoagulant activity and haemostatic efficacy in cuticle bleeding. These observations suggest that the heavy-chain sequence Lys713-Arg740, although dispensable for factor VIII cofactor function per se, is involved in the proteolytic activation of factor VIII both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1994

The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes.

Rates of factor X activation on endothelial cells were compared with activation rates on other vascular cells, platelets, monocytes and negatively charged phospholipid vesicles. Factor VIIa-mediated factor X activation was observed on smooth muscle cells and fibroblasts in the absence of cell-perturbing agents, whereas endothelial cells required activation in order to allow extrinsic activation of factor X. On the other hand, unperturbed endothelial cells did promote intrinsic, factor VIII/IXa-dependent activation of factor X. The rate of factor X activation on these cells was about one-sixth of that on ionophore A23187-stimulated platelets. Also, smooth muscle cells and fibroblasts were able to activate factor X through the intrinsic pathway, although to a lesser extent than endothelial cells. Monocytes were ineffective in this respect. Prothrombin fragment 1, the prothrombin fragment containing the gamma-carboxyglutamic acid domain known to mediate binding of vitamin K-dependent coagulation factors to phospholipid surfaces, inhibited factor VIII/IXa-dependent factor X activation on endothelial cells (IC50 3.2 microM) to a lesser extent than on phospholipid vesicles (IC50 0.2 microM). Therefore, besides negatively charged phospholipids, other membrane constituents seem to be involved in endothelial cell mediated, intrinsic activation of factor X. Perturbation of endothelial cells with phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) was without effect on intrinsic activation of factor X. This observation indicates that membrane constituents of endothelial cells involved in factor VIII/IXa-dependent activation of factor X are constitutively expressed.

Blood Coagulation↗
PubMed1982

The use of an activated factor IX complex (Autoplex) in the management of haemarthroses in haemophiliacs with antibodies to factor VIII.

A new activated factor IX product (Autoplex) has been used for the treatment of 18 haemarthroses in three haemophiliac patients with antibodies to factor VIII and a history of high anamnestic response to challenge with factor VIII. Results were evaluated by assessing pain, tenderness, range of movement and girth at 8 and 24 hours. A score of 2 was given for improvement at 8 hours and 1 for improvement at 24 hours. The result was expressed as a percentage of the possible score. Nine episodes treated with less than 35 units of Autoplex/kg had a mean score of 52% and nine episodes treated with more than 35 units/kg had a mean score of 73%. When only the more severe group of bleeds was considered, an even more marked dose related response emerged. When the ratio of the percentage fall in the PT to the percentage fall in the KCCT was plotted against the outcome, a significant correlation emerged (r = 0.43; P = 0.02).

Adolescent↗
PubMedDate not supplied

Prothrombin complex concentrates for clinical use.

Prothrombin complex concentrates prepared for clinical use are reviewed with emphasis on frequently observed adverse reactions such as viral hepatitis and thromboembolic complications. Preparation procedures and quality control are described briefly, and the use of these concentrates in the treatment of hemophilia A patients who develop factor VIII inhibitors is also described. The causes of and methods for the detection of potential thrombogenicity are discussed and suggestions which may result in a safer product are made.

Animals↗
PubMed1984

Non-A, non-B hepatitis in hemophilic patients with inhibitor treated with activated prothrombin complex concentrates: lack of correlation with an antigen possibly related to non-A, non-B hepatitis.

A population of 30 severe hemophilia-A patients with antibodies to factor VIII, treated with Autoplex since 1980, experienced a 30% incidence of non-A, non-B (NANB) hepatitis. 8 of the 9 patients affected had clinical signs of hepatitis and 7 had ALT levels in excess of 200 IU/l; the mean incubation time was 13 days. Only 5 of the 26 lots of Autoplex used were possibly transmitting the infective agent. An ELISA test to detect an antigen (DS-Ag) possibly related to NANB hepatitis was used to screen hemophilia-A and B patients. Its incidence was lower in patients treated less than 5 times a year (7.9%) than in patients treated over 15 times a year (25-27%) with locally prepared blood derivatives. Following treatment with Autoplex, the incidence of DS-Ag in inhibitor patients increased significantly (50%). In this last population, DS-Ag was shown to be unrelated to the NANB hepatitis observed. Although no direct evidence could be given, Autoplex was likely to transmit both the agent responsible for short incubation NANB hepatitis and DS-Ag.

Adolescent↗
PubMed2004

Recombinant human factor VIIa (rFVIIa) can activate factor FIX on activated platelets.

The studies reported here show that factor (F)VIIa can activate factor (F)IX on activated platelets in the absence of tissue factor. Both FIX and FIXa bind to the activated platelet surface with a K(d) of 8 nM and 2 nM, respectively. With factor (F)VIIIa, FIXa binds more tightly to platelets (K(d) 0.6 nM). At rFVIIa concentrations < 100 nm, no direct binding to the activated platelet surface can be detected with electrophoretic light scattering. However, in the presence of FIX, rFVIIa binding to platelets at concentrations as low as 10 nm rFVIIa can be detected. This is reflected by a decrease in the FIX K(d) from 8 to 1.6 nM. When rFVIIa is added to activated platelets in the presence of both FIX and FVIIIa, the K(d) for FIX decreases to 0.6, suggesting that rFVIIa activates FIX on the surface of activated platelets in the absence of tissue factor. The activation of FIX by FVIIa on activated platelets can also be demonstrated by a functional assay for FIXa. These data show that pharmacological doses of rFVIIa result in the direct activation of FIX by rFVIIa to form additional tenase complexes ultimately resulting in improved thrombin generation. These results may explain, at least in part, the mechanism of action of rFVIIa in hemorrhagic conditions seen in otherwise normal patients who develop an acquired coagulopathy due to trauma, surgery or a variety of other events in which rFVIIa has been found to be effective.

Blood Coagulation↗
PubMed2006

Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.

BACKGROUND: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. AIMS: Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII). METHODS: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. RESULTS: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. CONCLUSION: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.

Factor IXa↗
PubMed1980

Effects of low-dose 'factor VIII inhibitor bypassing activity (FEIBA)' in resistant haemophilia.

Possible effects and side-effects of 'factor VIII inhibitor bypassing activity (FEIBA)' were tested on 7 haemophiliacs with high-titre antibody to factor VIII (resistant haemophilia). FEIBA was administered both to hospitalised patients and as part of a home therapy programme. Serious side-effects, attributable to FEIBA, were one episode of hypersensitivity and, possibly, hepatitis. Our preliminary data suggest that early injection of FEIBA, using considerably lower doses than suggested by the manufacturer, may shorten the duration of immobilisation of haemophiliacs with inhibitor against factor VIII in comparison with infusion of factor VIII and/or supportive care alone, at least following musculoskeletal bleeding. A decrease or complete disappearance of the inhibitor was observed in all patients receiving FEIBA alone.

Adult↗