[The detection of Leiden mutation using technique of enzymatic extension of allele-specific primer with double bioluminescent detection (PED-Biolum)].

The article deals with comparing technique of detection of Leiden mutation on the basis of PEXT-reaction with subsequent bioluminescent microanalysis of products with technique based on RT-PCR. The sampling for testing comprised 83 specimen of genome DNA including 35 specimens with known Leiden heterozygote mutation. The commercial kit "SNP-express-PB" (Litex) was used as a comparison test. It is demonstrated that proposed approach is a simple in its application, effective and relatively inexpensive technique of detection of Leiden one-nucleotide polymorphism in gene V of blood coagulation factor. The technique "PED-Biolum" has no differences in comparison with commercial technique RT-PCR concerning ability to detect mutant allele and matches it in parameters of economic effectiveness.

Selective factor VIII and V inactivation by iminodiacetate ion exchange resin through metal ion adsorption.

The procoagulant activity of factors VIII and V depends on the presence of metal ion(s). We examined the effect of cation-exchange resins with different functional groups on both factors, of which only reaction with iminodiacetate resin resulted in the complete loss of their activity levels in plasma. However, the antigen level of factor VIII was preserved by >95%. This resin reduced divalent cations content present in factor VIII preparations, indicating that it inactivated this factor by direct deprivation of predominant Ca(2+) (>Mn(2+)>>Cu(2+)), rather than adsorption of the factor itself. The antigen level of recombinant factor VIII alone was decreased by >95% by reaction with resin, whilst that complexed with von Willebrand factor was preserved by >95%. Iminodiacetate resin-treated plasma was evaluated by measuring factor VIII and V activity in plasma with various levels of either activity. These were significantly correlated to the values obtained using factor VIII- or V-deficient plasma prepared commercially by immunodepletion. We demonstrated that iminodiacetate resin-induced factors VIII and V inactivation is because of direct deprivation of metal ions, predominantly Ca(2+), which is more essential for the functional structure of their molecules. Furthermore, iminodiacetate resin-treated plasma would be useful as a substrate for measuring the activity of these factors.

Separation of Factor V Leiden molecule, a mutated form of Factor V, from plasma of homozygous patient.

Factor V (FV) is a coagulant in plasma. The FV molecule consists of a heavy chain and a light chain, and Factor V Leiden (FVL) is mutated FV at a single amino acid in the heavy chain. FVL patients are in a dangerous hyper-coagulation state in their body. Current FVL diagnosis is done by DNA analysis, which is expensive and time consuming. Our group has been developing a real-time, cost effective immuno-optical biosensor for FVL diagnosis. For the sensor development, pure FVL, which is not currently available, is needed. Here, we have attempted FVL purification from FVL patient's plasma. Since plasma contains many proteins and some proteins are structurally homologous to FV, the purification must be done by a very specific method, such as immuno-affinity chromatography. However, an antibody that does not react with FV is not currently available. Because the mutation is in the heavy chain and the amino acid sequence of the light chain of FVL is identical to that of FV, antibodies generated against the light chain of FV were tested for purifying FVL. Plasma was obtained from a homozygous FVL patient. First, the plasma was pretreated by barium citrate and polyethylene glycol 6000, to remove the vitamin K-dependent proteins, alpha globulins, and other smaller than 6 kDa molecular weight proteins. The yield in the process was 54%. Immuno-affinity purification of FVL from patient plasma was then performed using an anti-FV light chain antibody immobilized CNBr-Sepharose, and the purification yield was 25%. In summary, the antibody against the light chain of FV was able to purify the single point mutated form of FV (FVL) from plasma with an overall yield of 14%. The same principle can probably be used for purification of the other single point mutated proteins.

QCM-D surpassing clinical standard for the dose administration of new oral anticoagulant in the patient of coagulation disorders.

The study focuses the dose administration of dabigatran to avoid the deaths due to hemorrhagic complications and thromboembolic stroke in clinics worldwide. To target the issue, a novel emerging acoustic technology, namely ''Quartz Crystal Microbalance with Dissipation'' (QCM-D) has been applied, while the acoustic assays namely ''activated Partial Thromboplastin Time'' (aPTT) and ''Prothrombinase complex-induced Clotting Test'' (PiCT) have been compared with the standard methods in parallel. Both techniques have been applied to 300 samples, including 220 plasma samples of patients suffering coagulation disorders and 80 plasma samples of non-patients. In comparison, the coagulation times of the acoustic aPTT and PiCT yielded an excellent correlation with the standard methods with in analytical standard deviation limits. Finally, the acoustic aPTT assay is the ''gold standard'' for a dose administration of the new oral anticoagulant, where the Δf/ΔΓ ratio of the acoustic assay demonstrates that dabigatran with FEIBA 50 combination could be a safe remedy to avoid the deaths in clinics.

Human coagluation factor V purification and thrombin-catalyzed activation.

Factor V was isolated from human plasma by barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B chromatography, ammonium sulfate fractionation, and gel chromatography on Ultrogel 22. Degradation of Factor V during purification was largely prevented by ample use of inhibitors of proteolytic enzyme. The purified Factor V was a stable, single-chain molecule with an apparent molecular weight of 330,000. Activation of human Factor V by thrombin resulted in a 10- to 15-fold increase in activity. The activation pattern as monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis was compared with that of bovine Factor V. Differences in the patterns of thrombin activation were noticed between the two species, whereas the final products were similar. The products of human Factor V activation are two closely spaced doublets, one with an apparent molecular weight of approximately 110,000, and the other, approximately 72,000. An antibody was raised against the purified protein. Crossed immunoelectrophoresis showed that the antibody recognized Factor V both before and after activation with thrombin.

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

Although platelets contain Factor V, localized primarily in the alpha-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chiu, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098 +/- 0.018 micrograms/10(5) cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234 +/- 0.180 micrograms/10(8) platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0 +/- 3.0 micrograms/ml) than that of Factor V antigen in human plasma (11.1 +/- 0.4 micrograms/ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01 +/- 1.09 micrograms/10(8) platelets) than do guinea pig were 2.85 +/- 0.30 U/ml plasma, 0.022 +/- 0.012 U/10(8) platelets, and 0.032 +/- 0.03 U/10(5) megakaryocytes, compared with human values of 0.98 +/- 0.02 U/ml plasma and 0.124 +/- 0.064 U/10(8) platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with [35S]methionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of 350,000. Both forms of Factor V were substrates for thrombin. Possible explanations for the higher molecular weight and different thrombin sensitivity and stability observed are that a precursor of Factor V was isolated or that the megakaryocyte Factor V had not been fully processed before isolation.

Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants.

Functional human Factor V has been purified using a rapid immunoaffinity method. Following barium citrate adsorption of plasma, Factor V was precipitated with polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized immunoadsorbent consisting of an IgG fraction containing a naturally occurring human monoclonal (IgG(4)lambda) antibody with inhibitory activity against human Factor V. The solid phase immunoglobulin quantitatively bound Factor V from human plasma. The bound Factor V was effectively eluted with a Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M alpha-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (M(r), 350,000) on both reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified Factor V when subjected to thrombin activation exhibited an 18-fold increase in coagulant activity. The isolated Factor V neutralized the inhibitory activities of the monoclonal antibody that was used to purify it, as well as the rabbit antibodies produced by immunizing the animals with the purified Factor V. Immunoelectrophoresis of purified Factor V against the polyclonal rabbit antiserum resulted in a single precipitin arc of identical mobility to the Factor V in normal human plasma. Analysis by double immunodiffusion showed a line of identity between plasma and purified Factor V and crossed immunoelectrophoresis showed a single species in normal plasma.A competitive enzyme-linked immunosorbent assay using the rabbit antibody against Factor V was applied to quantify Factor V antigen level in human plasma. Reconstitution of congenitally deficient or immunodepleted plasma with normal plasma or purified Factor V gave parallel dose-response curves. In 14 normal plasma the coagulant activity was 0.98+/-0.02 U/ml (mean+/-SEM) and antigen concentration was 11.1+/-0.4 mug/ml. A pool of 14 patients with congenital Factor V deficiency were studied. 10 patients had Factor V antigen ranging from 1.0 to 2.4 mug/ml with corresponding coagulant activities (0-0.17 U/ml) indicating a low concentration of normal Factor V, presumably due to decreased synthesis or increased degradation. When these patient plasmas and the normal plasmas were analyzed together an excellent correlation (r = 0.97, P < 0.01) was obtained. However, four patients with coagulant activity (0-0.08 U/ml) had Factor V antigen concentrations ranging from 4.4 to 6.1 mug/ml, indicating the presence of a reduced concentration of abnormal Factor V protein. The presence of patients with antigen similar in concentration to coagulant activity and antigen in excess of Factor V activity indicates the heterogeneity of congenital Factor V deficiency.

Isolation and partial characterization of human factor V.

Factor V was isolated from human citrate plasma by very mild purification steps. Cryoprecipitation, fractionation with polyethylene glycol 6000, gel filtration of AcA 44 and adsorption of haptoglobin to immobilized hemoglobin were applied successively, resulting in factor V preparations with a specific activity of 14.5 unit/mg. The yield was 28 percent. A molecular weight of 296 000 was determined by gel filtration and the apparent sedimentation constant found by ultracentrifugation in a sucrose gradient was 7.8 S. Parallel experiments with citrate plasma resulted in the same molecular weight and sedimentation constant. Polyacrylamide gel electrophoresis of factor V in the presence or absence of sodium dodecyl sulfate showed a single protein band. Incubation with human thrombin resulted in an 8-fold activation of the purified factor V.

The disociation of bovine factor V and isolation of the acitivated form.

Bovine Factor V disociated non-enzymatically to 276 000 dalton, whereas, the enzymatic activation produced Factor Va of 213 500 daltons. The disociated form, Factor Vd, was not an enzyme substrate for conversion to Factor Va. Factors V and Va sedimented in a density zone of 1.19 g/ml, and Factor Vd in 1.17 g/ml, in sucrose gradient centrifugation. Only a neutral lipid in a 7.2% yield could be extracted from Factor Va which had been isolated in a 4500-fold purification.

Identification of a contaminant protein in the purification of human factor V. Comparison of two purification methods.

Coagulation factor V has been isolated from human plasma by the methods of Bolhuis and of Kane to investigate reported differences in purity and specific activity. The specific activities of the preparations are 10 and 40 units/mg, respectively. The Kane preparation shows a single band (Mr 330 000) in polyacrylamide gel electrophoresis in the presence and absence of dithiothreitol, but the Bolhuis preparation shows an additional band at Mr 190 000 upon reduction with dithiothreitol. This suggests the presence of a contaminant which could account for the lower specific activity. Modification of the Bolhuis technique to include ion-exchange chromatography on DEAE-Sepharose resulted in separation of the two isolated proteins. The proteins were identified as factor V, with characteristics indistinguishable from Kane factor V, and a dimeric subunit of alpha 2-macroglobulin (Mr 350 000). Contamination by alpha 2-macroglobulin may explain the I2-component described in some preparations of bovine factor V, which is the basis of speculation that bovine factor V has a second polypeptide chain modulating stability and activity.

Assembly of prothrombinase complex.

The approaches described in this article have resulted in an increased understanding of the reaction steps involved in the stabilization and assembly of the prothrombinase complex. Because prothrombinase is considered an archetype for some of the other coagulation complexes, the quantitative information derived from these studies (Table I) provides the framework for future studies of prothrombinase and suggests experimental approaches for studies of the other analogous coagulation reactions.

Coagulation factor V: an old star shines again.

Blood coagulation factor V plays an important role in the regulation of thrombin formation. Activation of factor V by traces of activated coagulation factors (thrombin, factor Xa or meizothrombin) yields factor Va, the non-enzymatic cofactor of the prothrombinase complex. Since factor Va accelerates prothrombin activation under physiological conditions more than 10(4)-fold it is not surprising that down-regulation of factor Va cofactor activity by the protein C pathway is a very effective way for maintaining the hemostatic balance. In this paper we have reviewed the present status of structural knowledge of factor V and Va, the molecular changes in factor V that occur during factor V activation, the function of factor Va in prothrombin activation and the molecular mechanism of inactivation of factor Va by APC. Although considerable insight in the structure-function relationship of factor V and Va has been achieved, the study of mutated factor V molecules obtained by recombinant DNA technology will undoubtedly resolve remaining questions. The latter is illustrated by the fact that the discovery of factor VaLeiden has significantly contributed to our present knowledge on the regulation of the cofactor activity of factor Va via the protein C pathway. It appears that modulation of the activity of APC by protein S and factor Xa will strongly affect the in vivo activity of this pathway. Factor V not only plays an important role in the regulation of the activity of the prothrombinase complex but also acts as cofactor in APC-mediated inactivation of factor VIIIa. This gives rise to a rather intricate mechanism of regulation of thrombin formation by APC that thus far has been mainly studied in model systems containing purified proteins. Thus, extensive studies in plasma will be required in order to get more insight in the in vivo regulation of thrombin formation via the protein C pathway.

Expression and localization of tissue factor-based procoagulant activity (PCA) in pigeon monocyte-derived macrophages.

Monocyte-derived macrophages, focal to initiation and progression of atherosclerosis, have been implicated in thrombotic complication of this disease. In the present study we demonstrated tissue factor based procoagulant activity in cultured macrophages from the White Carneau pigeon following endotoxin (1-2 micrograms/ml) stimulation. This macrophage procoagulant activity paralleled activity obtained with pigeon brain homogenate. We used Enzyme-Linked Coagulation Assay (ELCA), an ultrasensitive microtiter plate assay, to measure procoagulant activity in these cells. Through the use of clotting factors purified from pigeon plasma, procoagulant activity could be detected with as few as 1-3 cells. Tissue factor antigen, detected through the use of immunogold labelling in conjunction with a polyclonal antibody which was highly specific to human tissue factor, was distributed uniformly over the plasma membrane of the endotoxin-stimulated cells. These studies suggest that this procoagulant activity might play an important role in the pathobiology of atherosclerosis in White Carneau pigeons by initiating fibrin polymerization and thus leading to thrombotic complications of the disease.

Characterization of monocyte-associated factor V.

We demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis. Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

An effect of predilution on VIII:C determination by the one-stage assay.

Factor VIII concentrates are usually prediluted in Owrens buffer, before use of the one-stage assay for VIII:C determinations. We found that predilution of high purity Factor VIII concentrates in fresh VIII:C deficient plasma gave VIII:C estimates about 3 times higher than predilution in Owrens buffer. Predilution in reconstituted lyophilized VIII:C deficient plasma gave VIII:C estimates about 1.6 times higher. Likewise the VIII:C in a plasma sample can be estimated to e.g. 0.03, 0.06 or 0.10 IU/ml depending on whether the reference curve is established by predilution of the reference plasma in fresh VIII:C deficient plasma, in reconstituted lyophilized VIII:C deficient plasma or in Owrens buffer, respectively. It is shown that the major part of the differences between the effects of the various prediluents on the VIII:C determinations, can be accounted for by differences in the content of Factor V, Fibrinogen, and vitamin K dependent coagulation factors in the assay mixtures.

The preparation and properties of factor V from bovine plasma.

1. A procedure for the purification of factor V from bovine plasma with a good yield and in a high state of purity is described. The isolation procedure involves BaSO4, adsorption, chromatography of the absorbed plasma on DEAE-Sephadex A-50, activation with thrombin of the A-50 eluate, chromatography on Amberlite CG-50 and precipitation with (NH4)2SO4. Factor V is obtained with a 160% yield and 5000-fold purification. 2. Some of the physico-chemical properties of Ac-globulin have been measured. The molecular weight was determined by electrophoresis is SDS-polyacrylamide gel and found to be 30 000. The studies employing disc electrophoresis and immunoelectrophoresis suggest the existence of several active isoforms of factor V.

A new model for coagulation factor V suggesting a unique mechanism of activation.

Blood coagulation factor V, the labile factor, is an important cofactor in the activation of prothrombin. Approximately 10 years ago, the first purification procedures for undegraded factor V from bovine and human plasma were reported. This was the starting point for a new area in the research on factor V structure-function relationships. In parallel to this, the structure of the even more labile anti-hemophilic factor (factor VIII) has been elucidated and the two proteins are found to be very similar in structure and in function. In this mini-review, I will focus on work performed in our laboratory, which has led forward to the proposal of a new structural model for factor V. It is based on results obtained with several different techniques, including protein chemistry, DNA technology and high resolution electron microscopy. In plasma, factor V circulates as a single chain, high molecular weight protein. During coagulation a limited number of peptide bonds are cleaved in the factor V molecule by thrombin. This leads to a great increase in biological activity. The active Va species is composed of a noncovalent complex between the N- and C-terminal fragments, whereas the activation fragments correspond to the carbohydrate-rich central portion of the molecule. The activity of factor Va is regulated through the selective degradation of the N-terminal heavy chain fragment by activated protein C. Purified human and bovine factor V was examined by high resolution transmission electron microscopy. Factor V was found to be composed of four major domains, three similar sized globular structures (diameter approx. 80 A) are linked via thin spacers to a larger central domain (diameter approx. 140 A). Activation with thrombin results in a reorganization of the molecule. The thrombin cleavage sites are positioned in the spacers between the different domains and two of the peripheral domains combine to form the active Va species. The new factor V model suggests that a unique and dramatic molecular reorganization occurs during the activation of factor V by thrombin and indicates that the low biological activity of single chain factor V is due to the physical separation of the N- and C-terminal domains by the large central region. Full biological activity can only be expressed after limited proteolysis by thrombin, when the two initially separated domains are free to combine to form the active factor Va molecule.

Studies on coagulation incompatibilities for xenotransplantation.

Microvascular thrombosis, following the activation of clotting cascade, is a hallmark of porcine solid organ xenograft rejection. The analysis of differences between human, monkey, and pig coagulation systems is crucial when monkey is used as animal model and pig as organ donor in xenotransplantation. Thrombosis, according to many authors, may be due to the molecular incompatibilities between natural anticoagulants present on pig endothelium and primate activated coagulation factors. The generation of activated protein C (PC) is critical for the physiological anticoagulation. One of the major incompatibilities may be related to the inability of pig thrombomodulin (TM) and endothelial protein C receptor to activate the recipient (primate) circulating PC in the presence of thrombin. Tissue factor pathway inhibitor (TFPI), is the primary inhibitor of tissue factor (TF)-induced coagulation. TFPI directly inhibits the activated factor X (FXa) and blocks the procoagulant activity of the TF/factor VIIa (FVIIa) complex by forming a quaternary TF/FVIIa/FXa/TFPI complex. Microvascular thrombosis, observed in the organ transplant, may also be due to the failure of pig TFPI to bind human FXa efficiently and inhibit human FVIIa/TF activity. The methods described in this chapter can be useful for the identification and characterization of primate and pig coagulation factors (isolated from a small volume of blood) by using SDS-PAGE and immunoblotting. Differences in molecular weight can help in the identification of the origin (pig or primate) of coagulation proteins in plasma from the recipient of xenografts. On the other hand, in vitro models of PC pathway and TFPI on human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) are described which can be used for studying incompatibilities between primate and pig.

Thrombosis risk in carriers of the factor V Leiden mutation: is it associated with a defined skin color?

Factor V Leiden (FVL; G1691A) is an autosomal dominant mutation with a high risk for thrombosis. Speculation that founders of FVL lived in the Middle East is supported by a prevalence of FVL that is higher in Arabs residing in Israel, Jordan, Lebanon, and Syria (12-14%) than in other white populations like Europeans (4-5%, up to 15% in the South of Sweden). We sought to verify the appropriate use of skin color as a clinical sign by which Arab individuals in Kuwait are included or excluded from testing for FVL. After institutional approval, 200 healthy Arabs residing in Kuwait consented to participate. Skin type was distinguished for the participants by Fitzpatrick natural skin color classification: 76 (38%) skin type II (white), 96 (48%) Mediterranean skin type IV (brown), and 28 (14%) skin type VI (black). FVL was tested by real-time PCR, and the percentage of carriers was calculated in each group. FVL was positive in 17 (8.5%) of the total subjects: 8 (10.5%) skin type II, 7 (7.3%) skin type IV, and 2 (7.1%) skin type VI. Therefore, FVL shows an even distribution in Arabs, and all Arabs residing in Kuwait should be tested for FVL irrespective of skin color.