Biomedical subjects

D M Monroe

Publications and source records attributed to D M Monroe.

At least 19 recordsLinked to original sources

PubMed2004

Role of the gamma-carboxyglutamic acid domain of activated factor X in the presence of calcium during inhibition by antithrombin-heparin.

BACKGROUND: Factor (F)Xa has 11 gamma-carboxylated glutamic acid (Gla) residues that are involved in calcium-dependent membrane binding. The serpin antithrombin (AT) is an important physiological regulator of FXa activity in an inhibition reaction that is enhanced by heparin. Recently, Rezaie showed that calcium further enhanced the heparin-catalyzed AT inhibition of FXa by promoting 'ternary complex' formation, and these results showed a role for the gamma-carboxyl-glutamate (Gla)-domain of FXa. OBJECTIVES: In this study, we used recombinant FXa mutants to assess the role of individual Gla residues in augmenting or antagonizing the AT-heparin inhibition reaction in the presence of calcium. RESULTS AND CONCLUSIONS: In the absence of heparin, AT inhibition of plasma and the recombinant FXas were essentially equivalent. Similar to plasma-derived FXa, calcium increased about 3-fold the inhibition rate of wild-type recombinant FXa by AT-heparin over that in the presence of EDTA. Interestingly, three different effects were found with the recombinant FXa Gla-mutants for AT-heparin inhibition: (i) Gla-->Asp 14 and 29 were enhanced without calcium; (ii) Gla-->Asp 16 and 26 were not enhanced by calcium; and (iii) Gla-->Asp 19 was essentially the same as wild-type recombinant FXa. These results support a theory that mutating individual Gla residues in FXa alters the calcium-induced conformational changes in the Gla region and affects the antithrombin-heparin inhibition reaction.

1-Carboxyglutamic Acid↗

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↗

PubMed2004

Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system.

Using a cell-based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0-200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV-deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell-based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin.

Blood Coagulation Factors↗

PubMed2001

The action of high-dose factor VIIa (FVIIa) in a cell-based model of hemostasis.

We have developed a cell-based model of hemostasis. This model suggests that the defect in hemophilia is specifically a failure of platelet-surface factor Xa (FXa) generation, leading to a failure of platelet surface thrombin generation. Activation of FX by FVIIa/tissue factor (TF) does not compensate for a lack of FXa activation on the platelet surface by the FVIIIa/FIXa complex. This is because plasma protease inhibitors prevent FXa from moving through the fluid phase from the TF-bearing cell to the platelet surface. We have previously proposed a platelet-dependent mechanism of action for high-dose factor VIIa (FVIIa; Novoseven, Novo Nordisk, Copenhagen, Denmark). Our data suggest that, when present at high levels, FVIIa binds to activated platelets and activates small amounts of FX independent of TF. This platelet-surface FXa can partially restore platelet-surface thrombin generation in hemophilia. Recently, van't Veer and colleagues reported results from an in vitro model in which coagulation reactions were initiated by relipidated TF. The authors concluded that high-dose FVIIa may exert a hemostatic effect in hemophilia by overcoming inhibition of FVIIa/TF activity by zymogen FVII. By contrast, we found that plasma levels of FVII did not slow thrombin generation in a model system initiated with cell-associated TF. This discrepancy highlights the potential differences between the studies of the coagulation reactions assembled on living cells compared to phospholipid vesicles. Our data suggest that in a cellular system high-dose FVIIa acts primarily by enhancing the rate of thrombin generation on platelet surfaces and not by overcoming inhibition by zymogen FVII of TF-dependent activation of FX.

Blood Coagulation↗

PubMed2001

High-dose factor VIIa increases initial thrombin generation and mediates faster platelet activation in thrombocytopenia-like conditions in a cell-based model system.

Clinical experience has shown that high doses of recombinant factor VIIa (rFVIIa) may ensure haemostasis in thrombocytopenic patients. We have used a cell-based model system to mimic thrombocytopenia and analyse the effect of rFVIIa. Lowering the platelet density from 200 x 10(9)/l (reflecting normal conditions) to 100, 50, 20 and 10 x 10(9)/l revealed a platelet density-dependent decrease in the maximal rate of thrombin generation, a prolongation in the time to maximal thrombin activity and a lower maximal level of thrombin formed. The platelet activation, measured as the time to half-maximal P-selectin (CD62) exposure, was not significantly dependent on the platelet density in the range of 200 x 10(9)/l to 10 x 10(9)/l, although there was a tendency for slower platelet activation at 20 x 10(9) and 10 x 10(9) platelets/l than at the higher platelet densities. Addition of 50--500 nmol/l rFVIIa to samples with 20 x 10(9) or 10 x 10(9) platelets/l shortened the lag phase of thrombin generation as well as the time to half-maximal platelet activation. Our data indicate that high doses of rFVIIa may help to provide haemostasis in thrombocytopenic patients by increasing the initial thrombin generation, resulting in faster platelet activation and thereby compensating for the lower number of platelets present.

Cells, Cultured↗

PubMed2001

A cell-based model of hemostasis.

Based on our work and that of many other workers, we have developed a model of coagulation in vivo. Many workers have demonstrated mechanisms by which cells can influence the coagulation process. Nonetheless, the prevailing view of hemostasis remains that the protein coagulation factors direct and control the process with cells serving primarily to provide a phosphatidylserine containing surface on which the procoagulant complexes are assembled. By contrast, we propose a model in which coagulation is regulated by properties of cell surfaces. This model emphasizes the importance of specific cellular receptors for the coagulation proteins. Thus, cells with similar phosphatidylserine content can play very different roles in hemostasis depending on their complement of surface receptors. We propose that coagulation occurs not as a "cascade", but in three overlapping stages: 1) initiation, which occurs on a tissue factor bearing cell; 2) amplification, in which platelets and cofactors are activated to set the stage for large scale thrombin generation; and 3) propagation, in which large amounts of thrombin are generated on the platelet surface. This cell based model explains some aspects of hemostasis that a protein-centric model does not.

Animals↗

PubMed2000

Deencryption of cellular tissue factor is independent of its cytoplasmic domain.

Tissue factor (TF) is a transmembrane molecule that, when exposed to plasma, is the key initiator of coagulation. Cellular TF activity is normally "encrypted", but treating cells with calcium ionophore (i.e. , ionomycin or A23187) increases ("deencrypts") TF activity without increasing TF mRNA or antigen expression. Deencryption results from both plasma membrane phosphatidylserine (PS)-dependent and -independent mechanisms; however, the nature of the PS-independent component is unclear. Since deencryption has been suggested to result from release of TF dimers on the cell surface, and since TF's cytoplasmic domain binds to actin-binding protein 280 and interacts with the cytoskeleton, we hypothesized that interactions with the cytoskeleton, through the cytoplasmic domain, play a role in mediating encryption/deencryption. We examined TF deencryption and the role of the cytoplasmic domain in the PS-independent component using baby hamster kidney (BHK) cells expressing full length TF (BHK-TF) or TF lacking its cytoplasmic domain (BHK-descyt) (Sorensen et al. (1999) J. Biol. Chem. 274, 21349). Both BHK-TF and BHK-descyt cells exhibited a dose-dependent, 1.5- to 10-fold increase in TF activity upon treatment with calcium ionophore, and this increase in activity was only partially blocked by annexin V. These results indicate that deencryption is not restricted to cells which naturally express TF and that the PS-independent component of deencryption is intact on cells transfected with either full length or truncated TF. Our results clearly indicate that deencryption is not dependent on an intact cytoplasmic domain in transfected BHK cells.

Animals↗

PubMed2000

Coagulation factor XI is a contaminant in intravenous immunoglobulin preparations.

A small number of thromboembolic events, including deep venous thrombosis and myocardial infarction, have been reported in patients receiving IVIG. These events have primarily occurred in patients receiving high-dose IVIG and have been attributed to an increase in blood viscosity. To test the hypothesis that a procoagulant might be present in IgG preparations, twenty-nine samples of intravenous immunoglobulin (IVIG) from eight different manufacturers were assayed for procoagulant activity. Twenty-six of these samples shortened the clotting time of factor XI-deficient plasma. Of these, fourteen samples had factor XI activities greater than 0.001 U/ml of normal pooled plasma. The remaining samples possessed less than 0. 001 U/ml of normal plasma activity. The procoagulant activity in these samples could be inhibited by an anti-factor XI polyclonal antibody, suggesting that the procoagulant activity was factor XI. The procoagulant activity increased in two samples after storage at 4 degrees C for 4 weeks, likely as a result of factor XIa autoactivation. Additionally, activity in some IVIG samples was able to directly activate factor IX, indicating that activated factor XI was present in these samples. Finally, the degree of factor XI(a) contamination in the samples was correlated with the manufacturer, suggesting that variations in the manufacturing process or source plasma affect the level of factor XI in the IVIG product. Because addition of small amounts of factor XIa to plasma can lead to production of significant amounts of thrombin, we suggest that factor XIa present in some IVIG preparations could contribute to the in vivo risk of thrombosis after IVIG therapy.

Drug Contamination↗

PubMed2000

The factor VII-platelet interplay: effectiveness of recombinant factor VIIa in the treatment of bleeding in severe thrombocytopathia.

Recently, high-dose factor VIIa has been used to correct bleeding in patients with various thrombocytopathias including Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and uremia. High-dose factor VIIa is postulated to act on platelets in the absence of tissue factor to activate factors IX and X and thus enhance thrombin generation. This enhanced thrombin generation might help provide hemostasis in patients with thrombocytopathias through several mechanisms. Enhanced thrombin generation would provide a strong signal for recruitment of other platelets. Also, enhanced fibrin deposition might provide mechanisms for bypassing the specific defect in thrombocytopathias. Thus, platelets from a patient with Bernard-Soulier syndrome might associate with fibrin by a glycoprotein IIb-IIIa-mediated mechanism. Also, platelets from a patient with Glanzmann's thrombasthenia might associate with fibrin through von Willebrand factor-mediated interactions with glycoprotein Ib-V-IX. Finally, enhanced thrombin generation on platelets would mean that fewer platelets are required for hemostasis.

Blood Platelet Disorders↗

PubMed2000

The effect of factor X level on thrombin generation and the procoagulant effect of activated factor VII in a cell-based model of coagulation.

We used a cell-based, in-vitro model of normal hemostasis and hemophilia to address the question of whether factor (F) X concentration affects the hemostatic response to high-dose activated factor VII (FVIIa). Under conditions designed to mimic normal tissue factor-initiated hemostasis in vivo, we found that only a very small amount of FX -- equivalent to about 3% of the normal plasma level -- was required to support a 'normal' level of thrombin generation. This suggests that, under normal conditions in vivo, the level of FX does not significantly affect hemostatic function. By contrast, in experiments designed to mimic the hemophilic condition, the level of FX had a significant effect on the level of thrombin generated in the presence of high-dose FVIIa. This finding suggests that the plasma level of FX could affect the hemostatic response of hemophilic patients to high-dose FVIIa therapy.

Blood Coagulation↗

PubMed2000

Links between the immune and coagulation systems: how do "antiphospholipid antibodies" cause thrombosis?

Inflammation and immune activation have been associated with thrombosis in a number of settings. We have been interested in the question of how the presence of a type of autoantibody, so-called "antiphospholipid" antibody, leads to thrombosis. Several mechanisms have been proposed including modulation of tissue factor expression, enhancement of procoagulant binding to platelets, and interference with antithrombotic mechanisms. We developed a cell-based model of coagulation that, unlike current coagulation assays, reflects some of the in vivo activities of "antiphospholipid" antibodies. "Antiphospholipid" antibodies against the phospholipid-binding protein beta-2-glycoprotein-1 enhance thrombin generation in this model system, primarily by enhancing procoagulant reactions on tissue factor-bearing cells.

Antibodies, Antiphospholipid↗

PubMed1999

Thrombin activates factor XI on activated platelets in the absence of factor XII.

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.

Anticoagulants↗

PubMed1999

Tissue factor de-encryption: ionophore treatment induces changes in tissue factor activity by phosphatidylserine-dependent and -independent mechanisms.

Coagulation is initiated on tissue-factor-bearing cells when factor VIIa complexes with membrane-bound tissue factor and activates factors X and IX. Cellular tissue factor activity does not correlate with tissue factor antigen; treatment with calcium ionophore rapidly increases tissue factor activity without increasing tissue factor antigen. Our study examined the effect of calcium ionophore A23187 on tissue factor activity of freshly isolated, lipopolysaccharide-stimulated monocytes and non-transformed human dermal fibroblasts. A23187 increased tissue factor activity on monocytes and fibroblasts in a dose-dependent fashion between 0.1 and 50 micromol/l ionophore. This increase in activity was proportional to an increase in intracellular calcium in monocytes. The increase in tissue factor activity was partially attributable to an increase in phosphatidylserine expression, as measured by increased prothrombinase activity (1.1- to 4-fold) on ionophore-treated cells. The phosphatidylserine-binding protein annexin V decreased tissue factor activity on both ionophore-treated and untreated cells, reflecting the role of phosphatidylserine in tissue factor activity. However, even in the presence of saturating concentrations of annexin V, the tissue factor activity of ionophore-treated cells was 1.3- to 11.3-fold higher than that of untreated cells, indicating that the increase in tissue factor activity did not result solely from increased expression of phosphatidylserine. A23187 increased tissue-factor-dependent activation of factors IX and X 1.4- to 7-fold on both cell types, indicating that ionophore treatment did not alter factor VIIa/tissue factor substrate specificity. We conclude that the mechanism by which calcium ionophore increases tissue factor activity is not unique to monocytoid or transformed cells. Furthermore, the ionophore-induced increase in activity is not solely the result of increased exposure to phosphatidylserine. Finally, tissue factor de-encryption by A23187 does not alter factor VIIa/tissue factor substrate specificity.

Calcimycin↗

PubMed1999

TFPIbeta, a second product from the mouse tissue factor pathway inhibitor (TFPI) gene.

Tissue factor pathway inhibitor (TFPI) contains three Kunitz domains separated by two connecting regions. We have cloned another naturally occurring TFPI gene product from a mouse lung cDNA library which we have called TFPIbeta. TFPIbeta is derived from alternative splicing of the TFPI gene. Analysis of the cDNA shows that mouse TFPIbeta protein is identical to TFPI from the N'-terminus through the second connecting region. However, mouse TFPIbeta possesses neither a third Kunitz domain nor an Arg, Lys-rich C'-terminus but instead has a completely different C'-terminal (beta-domain) sequence which is not homologous to any known protein. Northern blot analyses show that the tissues for mouse TFPIbeta synthesis are heart and lung; in contrast, TFPI appears in Northern blots of heart and spleen. Both TFPIbeta and TFPI messages first appear in 7-day-old mouse embryos, but only the TFPI mRNA persists until 17 days. Purified recombinant TFPIbeta shows an apparent molecular weight of 38 kDa. Kinetic studies indicate that mouse TFPIbeta is a slow-binding enzyme inhibitor for human factor Xa. In addition, heparin does not enhance the inhibition of factor Xa by mouse TFPIbeta although it does accelerate factor Xa inhibition by TFPI.

Amino Acid Sequence↗

PubMed1998

Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles.

This report describes the expression, purification, and characterization of a series of recombinant factor Xa variants bearing aspartate substitutions for each of the glutamate residues which normally undergo gamma-carboxylation. Factor X was expressed in human embryonic kidney cells and purified from conditioned media by immunoaffinity and hydroxylapatite chromatography. Factor X was activated with Russell's viper venom factor X activator, and single-chain unactivated factor X was removed from activated factor X by size-exclusion chromatography. Recombinant wild-type factor Xa had normal activity in a clotting assay, and mutants with aspartate substitutions for glas residues 16, 26, and 29 had no detectable clotting activity. In purified component assays, these gla variants had essentially no detectable activity in the prothrombinase complex assembled on synthetic phospholipid vesicles but had significant activity when the prothrombinase was assembled on thrombin-activated platelets. In addition, the gla 32 variant had normal activity in the platelet prothrombinase but diminished activity in prothrombinase assembled on synthetic PSPC vesicles. These differences were not accounted for by the total phospholipid composition of the thrombin-activated platelet membrane. We have produced fully active recombinant human factor Xa and demonstrated that gla residues 16, 26, and 29 are critical for normal activity of factor Xa. More importantly, this study provides an extensive characterization of macromolecular enzyme complex formation with gla variants of a vitamin K-dependent coagulation protein and provides evidence that prothrombinase complex assembly on thrombin-activated platelets is not equivalent to assembly on synthetic phospholipid vesicles. The data suggest that thrombin-activated platelets possess some element(s) (other than 30% phosphatidyl serine or factor Va), presumably either protein or phospholipid, that serves as a component of the factor Xa binding site.

Blood Coagulation↗

PubMed1998

Newer concepts of blood coagulation.

In this report we describe an in vitro model of blood coagulation reactions that mimics as closely as possible the in vivo condition. Our model indicates that the tissue factor-factor VIIa complex initiates coagulation by activating small amounts of both factor IX and factor X in the environment of the tissue factor bearing cell. Factor Xa and factor IXa formed in the initial reaction then play very distinct roles in the subsequent interactions of the clotting mechanism leading to a burst of thrombin generation on the platelet surface. Our results also indicate that factor XI can be activated by thrombin in the absence of factor XII and that the function of factor XI is simply to enhance conversion of factor IX to factor IXa resulting in enhanced thrombin generation on the platelet surface.

Animals↗

PubMed1998

Cloning, expression, and characterization of mouse tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

Amino Acid Sequence↗

PubMed1998

Active site-inactivated factors VIIa, Xa, and IXa inhibit individual steps in a cell-based model of tissue factor-initiated coagulation.

Factors VIIa, Xa, and IXa play different roles in the initiation of tissue factor-dependent coagulation. The consequences of competing with the different enzymes were investigated, thereby examining the effects of inhibiting the initiation process at different steps. Active site-inactivated factors VIIa, Xa, and IXa (FVIIai, FXai, and FIXai, respectively) were added to various cell-based assays mimicking the individual steps in tissue factor-initiated coagulation. In an assay involving tissue factor-expressing monocytes, coagulation proteins and unactivated platelets, FVIIai and FXai inhibited platelet activation and thrombin generation while FIXai only inhibited thrombin generation. FVIIai inhibited factor Xa generation and subsequent thrombin generation on monocytes, while FXai inhibited thrombin generation on the monocytes as well as on the activated platelets. FIXai had no effect on factor Xa or thrombin generation on the monocytes, but inhibited factor Xa and subsequent thrombin generation on the activated platelets. FVIIai had no effect on the reactions taking place on the activated platelets. The data confirm a model where tissue factor/factor VIIa mediates factor Xa generation and subsequent prothrombin activation on the tissue factor-bearing cells. Thrombin then activates platelets, which serve as the physiologically important surface for large-scale thrombin generation.

Adult↗