Development of antitissue factor antibodies in patients after liver surgery

After liver surgery, two patients developed unexplainable prolonged prothrombin times (PT) that were not associated with bleeding tendency. The substitution of rabbit thromboplastin for either human or monkey thromboplastin in performing PT tests resulted in a normal clotting time. Tissue factor (TF) procoagulant activity assays and an immunoblotting analysis showed that these patients had developed IgG lambda-type immediate anticoagulants directed against both rabbit and bovine TF that did not crossreact with human or monkey TF. In a chromogenic assay, the patient IgG caused a decrease in both the Km and the Vmax of the factor X activation by rabbit TF-factor VIIa complex. The lack of reactivity of the patient IgG with human TF presumably explained why there was no clinical bleeding. Both patients had been treated earlier with a topical hemostatic agent prepared from bovine corium, microfibrillar collagen hemostat, while undergoing previous surgery. In an immunoblotting analysis, the patient IgG stained a 42-Kd band in the Triton extract of the collagen preparation under either reducing or nonreducing conditions. The Triton extract of the collagen preparation blocked the binding of the patient IgG to bovine TF. Thus, it is suggested that the iatrogenic immunization by intraoperative exposure of bovine TF retained in the collagen preparation may be responsible for the development of anti-TF antibodies in these patients. The anti-TF antibodies resulted in a clinical error in the evaluation of coagulation status after the use of rabbit thromboplastin.

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A soluble tissue factor-annexin V chimeric protein has both procoagulant and anticoagulant properties

Blood ◽

10.1182/blood-2005-07-2733 ◽

2006 ◽

Vol 107 (3) ◽

pp. 980-986 ◽

Cited By ~ 13

Author(s):

Xin Huang ◽

Wei-Qun Ding ◽

Joshua L. Vaught ◽

Roman F. Wolf ◽

James H. Morrissey ◽

...

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Vascular Injury ◽

Factor Viia ◽

Chimeric Protein ◽

Annexin V ◽

Factor X ◽

Low Concentrations ◽

Finite Period ◽

Factor X Activation

AbstractTissue factor (TF) initiates blood coagulation, but its expression in the vascular space requires a finite period of time. We hypothesized that targeting exogenous tissue factor to sites of vascular injury could lead to accelerated hemostasis. Since phosphatidylserine (PS) is exposed on activated cells at sites of vascular injury, we cloned the cDNA for a chimeric protein consisting of the extracellular domain of TF (called soluble TF or sTF) and annexin V, a human PS-binding protein. Both the sTF and annexin V domains had ligand-binding activities consistent with their native counterparts, and the chimera accelerated factor X activation by factor VIIa. The chimera exhibited biphasic effects upon blood coagulation. At low concentrations it accelerated blood coagulation, while at higher concentrations it acted as an anticoagulant. The chimera accelerated coagulation in the presence of either unfractionated or low-molecular-weight heparins more potently than factor VIIa and shortened the bleeding time of mice treated with enoxaparin. The sTF-annexin V chimera is a targeted procoagulant protein that may be useful in accelerating thrombin generation where PS is exposed to the vasculature, such as may occur at sites of vascular injury or within the vasculature of tumors.

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Protein disulfide isomerase has no stimulatory chaperone effect on factor X activation by factor VIIa-soluble tissue factor

Thrombosis Research ◽

10.1016/j.thromres.2008.04.012 ◽

2008 ◽

Vol 123 (1) ◽

pp. 171-176 ◽

Cited By ~ 16

Author(s):

Egon Persson

Keyword(s):

Tissue Factor ◽

Protein Disulfide Isomerase ◽

Factor Viia ◽

Factor X ◽

Disulfide Isomerase ◽

Factor X Activation ◽

Protein Disulfide

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Analysis Of The Kinetics Of Factor X Activation By Tissue Factor-Factor VIIa

Pathophysiology of Haemostasis and Thrombosis ◽

10.1159/000217290 ◽

1996 ◽

Vol 26 (4) ◽

pp. 98-101 ◽

Cited By ~ 1

Author(s):

Yale Nemerson

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Factor X ◽

Factor X Activation ◽

Kinetics Of

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The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation

Blood ◽

10.1182/blood.v65.5.1226.1226 ◽

1985 ◽

Vol 65 (5) ◽

pp. 1226-1231 ◽

Cited By ~ 16

Author(s):

TB McNeely ◽

MJ Griffith

Keyword(s):

Antithrombin Iii ◽

Factor Xa ◽

Coagulation Factors ◽

Factor Ix ◽

Factor X ◽

Clotting Time ◽

Intrinsic Pathway ◽

Blood Coagulation Factors ◽

Factor Ixa ◽

Factor X Activation

Abstract The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

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Factor VIIa-catalyzed activation of factor X independent of tissue factor: its possible significance for control of hemophilic bleeding by infused factor VIIa

Blood ◽

10.1182/blood.v75.5.1069.1069 ◽

1990 ◽

Vol 75 (5) ◽

pp. 1069-1073 ◽

Cited By ~ 62

Author(s):

LV Rao ◽

SI Rapaport

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Activate Factor ◽

Factor X ◽

Extrinsic Pathway ◽

Hemophilic Patient ◽

Factor X Activation ◽

Rate Of Activation

Abstract Infusing factor VIIa (FVIIa) has been reported to control bleeding in hemophilic patients with factor VIII (FVIII) inhibitors. This is difficult to attribute to an enhanced FVIIa/tissue factor (TF) activation of factor X, since in vitro studies suggest that infusion of FVIIa should neither increase substantially the rate of formation of FVIIa/TF complexes during hemostasis (Proc Natl Acad Sci USA 85:6687, 1988) nor bypass the dampening of TF-dependent coagulation by the extrinsic pathway inhibitor (EPI) (Blood 73:359, 1989). Partial thromboplastin times have also been reported to shorten after infusion of FVIIa. The experiments reported herein establish that shortening of partial thromboplastin times after adding FVIIa to hemophilic plasma in vitro stems from an FVIIa-catalyzed activation of factor X independent of possible trace contamination of reagents with TF. Experiments in purified systems confirmed that FVIIa can slowly activate factor X in a reaction mixture containing Ca2+ and phospholipid but no source of TF. The rate of activation was sufficient to account for the shortening of partial thromboplastin times observed. EPI, which turned off continuing FVIIa/TF activation of factor X, was unable to prevent continuing FVIIa/phospholipid activation of factor X. Because circulating plasma contains only a trace, if any, free FVIIa, such a reaction could never occur physiologically. However, infusing FVIIa creates a nonphysiologic circumstance in which a continuing slow FVIIa/phospholipid catalyzed activation of factor X could conceivably proceed in vivo unimpeded by EPI. Such a mechanism of factor X activation might compensate for an impaired factor IXa/FVIIIa/phospholipid activation of factor X during hemostatis, and therefore control bleeding in a hemophilic patient.

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Extrinsic Activation of Human Blood Coagulation Factors IX and X

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645199 ◽

1990 ◽

Vol 63 (02) ◽

pp. 224-230 ◽

Cited By ~ 11

Author(s):

V J J Bom ◽

J H Reinalda-Poot ◽

R Cupers ◽

R M Bertina

Keyword(s):

Tissue Factor ◽

Kinetic Data ◽

Factor Viia ◽

Coagulation Factors ◽

Factor Ix ◽

Lag Phase ◽

Factor X ◽

Extrinsic Factor ◽

Factor Ixa ◽

Factor X Activation

SummaryWe studied activation of human coagulation factors IX and X by factor VIIa in the presence of calcium ions, phospholipid (phosphatidylserine/phosphatidylcholine, 50/50, mol/mol) and purified tissue factor apoprotein. Activation of factor IX and factor X was found to occur without a measurable lag-phase and hence initial rates of factor IXa and factor Xa formation could be determined. Like previously observed for the activation of factor X, the activation of factor IX was saturable with respect to factor VIIa, tissue factor apoprotein and phospholipid. The results suggested that in the presence of a Ca2+ ions the same ternary complex of factor VIIa-tissue factor apoprolein-phospholipid is responsible for the activation of factor IX and factor X. Roth the apparent Km of 22 nM-factor IX and the apparent Kcat of 28 min−1 were about 3-fold lower than the coiicsponding parameters of factor X activation by this complex. Hence, the catalytic efficiency (Kcat/Km) of factor IX and factor X activation was about equal. However, the two substrates inhibited the activation of each other by competition for the same catalytic sites. The apparent Kinh of factor IX for inhibition of extrinsic factor X activation is 30 nM. The apparent Kinh of factor X for inhibition of extrinsic factor IX activation is 116 nM. From these kinetic data it was calculated that at plasma concentration of factors IX and X, the rate of extrinsic factor IX activation would be half the rate of factor X activation. These relative rates of extrinsic factor IX and factor X activation in combination with previously reported kinetic data on the activation of factor X by factor IXa in the presence of factor VIIIa provide support for the concept that at low levels of tissue factor, factor IXa formation might play an important role in the extiinsic pathway of coagulation in vivo.

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Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615835 ◽

1999 ◽

Vol 82 (08) ◽

pp. 209-217 ◽

Cited By ~ 36

Author(s):

Patrick Celie ◽

Joost Kolkman ◽

Peter Lenting ◽

Koen Mertens

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Factor Viia ◽

Three Dimensional ◽

Factor Ix ◽

Coagulation Cascade ◽

Factor X ◽

Factor Ixa ◽

Factor Viiia ◽

Factor X Activation

IntroductionThe activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.

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Murine Model of Suppressed Intrinsic Pathway Using a FX Variant: FX Roma.

Blood ◽

10.1182/blood.v106.11.728.728 ◽

2005 ◽

Vol 106 (11) ◽

pp. 728-728

Author(s):

Paris Margaritis ◽

Shing Jen Tai ◽

Rodney M. Camire ◽

Danielle Dunn ◽

Katherine A. High

Keyword(s):

Factor Viia ◽

Es Cells ◽

Site Directed Mutagenesis ◽

Intrinsic Activity ◽

Bleeding Tendency ◽

Factor X ◽

Intrinsic Pathway ◽

Hprt Gene ◽

Extrinsic Pathway ◽

Coagulation Testing

Abstract The complex interplay between the intrinsic and extrinsic pathways of coagulation is incompletely understood. The existence of Factor X variants that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma, De Stefano et al., Br J Haematol.1988, 69(3):387–91), is due to a missense mutation (Thr318→Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3%) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50%) in the extrinsic pathway as measured by PT. Using site-directed mutagenesis and transient transfection in HEK 293 cells, we confirmed this differential activity in extrinsic and intrinsic pathways for a variant human Factor X (FX) recombinant protein with a Thr318→Met change. FX antigen was assessed by ELISA as previously described (Larson PJ et al., Biochemistry.1998, 37(14):5029–38) and FX activity by PT or aPTT; analysis of conditioned medium from FX wild-type and FX Thr318→Met showed respectively: antigen 100%, 82%; intrinsic activity 120 U/mg (100%), 1.8 U/mg (1.5%); extrinsic activity 190 U/mg (100%), 81 U/mg (41%). To further study this variant, we constructed a mouse homozygous for the analogous mutation (Thr318→Met) in the murine FX gene. This residue is conserved in human, murine and canine FX. We utilized mouse ES cells in which exon 8 of the murine FX gene had previously been deleted and replaced with a neomycin resistance gene and a partial HPRT gene through a targeted recombination event. These cells were used for a second electroporation event with a targeting construct carrying an exon 8 cassette containing the Thr318→Met substitution, and the missing portion of the HPRT gene. After selection on HAT medium, correctly targeted ES cells were micro-injected into blastocysts and implanted into pseudopregnant mice to generate chimeric mice. Heterozygous offspring of chimeric males were intercrossed to obtain animals homozygous for the FXRoma mutation. Coagulation testing revealed that FXRoma heterozygotes showed the expected 52.1±5.9% intrinsic activity and 142.8±25.1% extrinsic activity. More importantly, FXRoma homozygotes showed 8.5±2.7% intrinsic activity and 107.7±41.1% extrinsic activity. Thus the murine FXRoma mutation demonstrates a coagulation profile similar to that observed with the human variant. Thus the FXRoma mouse can model the effect of impeded intrinsic pathway more accurately than e.g. a FIX knockout mouse, because both FX and FIX can act as substrates for the Factor VIIa-Tissue Factor complex and all the components of the Xase complex will be essentially at normal or near-normal levels. The ability to carry out studies of long-term effects of an impeded pathway, and to analyze the response of these animals to prothrombotic stimuli, will allow us to determine the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway.

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Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid

Blood ◽

10.1182/blood.2020005930 ◽

2020 ◽

Vol 136 (21) ◽

pp. 2469-2472 ◽

Cited By ~ 2

Author(s):

Valerie A. Novakovic ◽

Gary E. Gilbert

Keyword(s):

Protein C ◽

Factor Viia ◽

Activated Protein C ◽

Factor X ◽

Cardiac Myosin ◽

Prothrombinase Complex ◽

Skeletal Myosin ◽

Factor X Activation ◽

Muscle Myosin ◽

Prothrombinase Activity

Abstract Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine–binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

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