Functional Effects of Anticardiolipin Antibodies

The ‘lupus anticoagulant’ phenomenon is the best documented functional effect of antiphospholipid (aPL) antibodies, occurring either by inhibition of the prothrombinase and/or Factor X activation reactions. Understanding the mechanism by which aPL antibodies inhibit phospholipid dependent coagulation reactions may yield important clues about their ‘thrombogenic effects’ in vivo. We conducted a series of studies to determine the specificity, diversity, and mechanism by which aPL antibodies inhibit phospholipid dependent reactions. Results showed that purified immunoglobulins with lupus anticoagulant and anti-cardiolipin activities were absorbed by negatively charged phospholipids and both activities were recovered from the phospholipid-antibody precipitate. Purified aPL antibodies inhibited the prothrombinase reaction in a plasma free system in which β2-glycoprotein 1 (β2-GP1) was absent. Affinity purified aPL antibodies had 25–50 times the inhibitory activity of immunoglobulin preparations. The phospholipid binding proteins, β2-GP1 and placental anticoagulant protein I (PAP I), independently inhibited the prothrombinase reaction, and when these proteins were combined with aPL, inhibition of the prothrombinase reaction was additive. Antibodies of syphilis had no inhibitory effect, partially accounted for by lack of specificity for phosphotidylserine (PS). Although aPL antibodies inhibited the protein C activation reaction, there was no correlation of these activities with inhibition of the prothrombinase reaction. Together, these results show that aPL exert their effects by interaction with negatively charged phospholipids, in particular phosphotidylserine, but lack of correlation between inhibition of the prothrombinase and protein C activation reactions, suggests that the nature of the coagulation protein is also important.

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Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Blood ◽

10.1182/blood.v86.3.1062.bloodjournal8631062 ◽

1995 ◽

Vol 86 (3) ◽

pp. 1062-1071 ◽

Cited By ~ 2

Author(s):

SJ Koppelman ◽

TM Hackeng ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Factor Viii ◽

Protein C ◽

Intrinsic Factor ◽

Activated Protein C ◽

Protein S ◽

Inhibitory Effect ◽

Factor X ◽

Ic50 Values ◽

Factor X Activation ◽

Factor X Activating Complex

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

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Functional and binding studies of the roles of prothrombin and beta 2- glycoprotein I in the expression of lupus anticoagulant activity

Blood ◽

10.1182/blood.v83.10.2878.2878 ◽

1994 ◽

Vol 83 (10) ◽

pp. 2878-2892 ◽

Cited By ~ 93

Author(s):

P Permpikul ◽

LV Rao ◽

SI Rapaport

Keyword(s):

Calcium Ions ◽

Lupus Anticoagulant ◽

Anticoagulant Activity ◽

Protein A ◽

Inhibitory Effect ◽

Factor X ◽

Glycoprotein I ◽

Normal Plasma ◽

Beta 2 ◽

Factor X Activation

Abstract We present functional and binding data relevant to the reported roles for prothrombin and beta 2-glycoprotein I (beta 2GPI) in the expression of lupus anticoagulant activity. In a purified system containing human prothrombin, Xa, Va, and a rate-limiting concentration of phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles, the preliminary incubation of vesicles with protein A separated IgG preparations from 10 lupus anticoagulant plasmas, calcium, and prothrombin enhanced the inhibitory effect of all IgG preparations upon thrombin generation. Experiments in a purified factor X activation system provided supporting data that a similar preliminary incubation with prothrombin enhanced the inhibitory effect of many of the IgG preparations upon factor X activation. However, we could not obtain unequivocal evidence that prothrombin was an obligatory cofactor for lupus anticoagulant IgG to inhibit procoagulant phospholipid function, because lupus anticoagulant IgG separated by protein A chromatography contained traces of prothrombin. The binding of many IgG preparations to immobilized PS was enhanced by prothrombin when calcium ions were present. beta 2GPI enhanced binding of many of the IgG preparations to immobilized PS both in the presence and absence of calcium, yet beta 2GPI failed to enhance the ability of the IgG preparations to inhibit phospholipid function in purified prothrombin and factor X assays. Moreover, the IgG preparations prolonged the dilute Russell's viper venom time (dRVVT) of beta 2GPI-depleted normal plasma. Nine of 10 IgG preparations bound to prothrombin on Western blots in the absence of calcium and phospholipid, whereas no preparation bound to beta 2GPI. Passage of five citrated lupus anticoagulant plasmas through a prothrombin affinity column in the absence of added calcium and phospholipid removed most of the activity prolonging the dRVVT of normal plasma, and IgG in the pass-through plasma no longer bound to PS in the presence of prothrombin and calcium ions. IgG in prothrombin column eluates had strikingly enhanced specific lupus anticoagulant activity and also specific PS binding activity in the presence of prothrombin and calcium ions. Thus, lupus anticoagulant plasmas were shown to contain IgG binding to prothrombin, in the absence of calcium ions and phospholipid, which could also, in the presence of calcium ions and prothrombin, bind to PS and express lupus anticoagulant activity.

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Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Blood ◽

10.1182/blood.v86.3.1062.1062 ◽

1995 ◽

Vol 86 (3) ◽

pp. 1062-1071 ◽

Cited By ~ 45

Author(s):

SJ Koppelman ◽

TM Hackeng ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Factor Viii ◽

Protein C ◽

Intrinsic Factor ◽

Activated Protein C ◽

Protein S ◽

Inhibitory Effect ◽

Factor X ◽

Ic50 Values ◽

Factor X Activation ◽

Factor X Activating Complex

Abstract Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

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Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

Blood ◽

10.1182/blood.v86.7.2653.2653 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2653-2660 ◽

Cited By ~ 17

Author(s):

SJ Koppelman ◽

C van't Veer ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Monoclonal Antibodies ◽

Factor Viii ◽

Protein C ◽

Binding Protein ◽

Intrinsic Factor ◽

Protein S ◽

Inhibitory Effect ◽

Factor X ◽

Beta Chain ◽

Factor X Activation

Abstract The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

Blood ◽

10.1182/blood.v86.7.2653.bloodjournal8672653 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2653-2660

Author(s):

SJ Koppelman ◽

C van't Veer ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Monoclonal Antibodies ◽

Factor Viii ◽

Protein C ◽

Binding Protein ◽

Intrinsic Factor ◽

Protein S ◽

Inhibitory Effect ◽

Factor X ◽

Beta Chain ◽

Factor X Activation

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5670 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5670-5677 ◽

Cited By ~ 82

Author(s):

Ossama Abu Hatoum ◽

Shlomit Gross-Mesilaty ◽

Kristin Breitschopf ◽

Aviad Hoffman ◽

Hedva Gonen ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Specific Inhibitor ◽

Inhibitory Effect ◽

26S Proteasome ◽

Intact Cells ◽

Free System ◽

Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

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THE "LUPUS ANTICOAGULANT" INDUCES FUNCTIONAL CHANGES IN ENDOTHELIAL CELLS AND PLATELETS

10.1055/s-0038-1643656 ◽

1987 ◽

Author(s):

Anna E Schorer ◽

Kathleen V Watson

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Lupus Anticoagulant ◽

Inhibitory Effect ◽

Clinical Syndrome ◽

Factor Vii ◽

Factor X ◽

Functional Changes ◽

Thrombin Activation

The presence of the "lupus anticoagulant" (LA) predicts a clinical syndrome of excessive arterial, venous and microvascu-lar thrombosis. LA is an antibody which reacts with negatively charged phospholipid (PL) species in vitro. Since PL is involved in many aspects of the regulation of thrombosis, we postulated that LA might modify one or more of the membrane-(PL-dependent reactions of platelets and endothelial cells (EC). Blood samples from 20 patients with a history of thrombosis were tested for the presence of LA (kaolin PTT) and titres determined. LA-positive (LA+) sera and plasma were compared to LA-negative (LA−) samples from normal donors (n=6) or patients who had lupus but no clinical thrombosis (n=4). These specimens were tested in a panel of assays. The thrombin-stimulated release of prostacyclin (PG12) from cultured human EC was markedly reduced (52%±12.5 s.e.) by preincubation of the EC with LA+ sera (30 minutes). Purified LA+ IgG from one patient reproduced this effect. Thrombin induction of EC synthesis of the procoagulant, tissue factor-which is dissociable from prostaglandin metabolism-was also inhibited by LA+ sera. Normal platelets incubated in LA+ plasma became refractory to thrombin (1 unit/ml) but retained their responsiveness to epinephrine and ADP. The reduced responsiveness to thrombin was not due to altered (specific or total) binding of thrombin. The cleavage of Factor X by Factor VII requires PL as a co-factor for the EC procoagulant, tissue factor (TF). Unlike the inhibitory effect of LA on thrombin activation of EC and platelets, this distinct membrane-(PL-) dependent function was variably enhanced by LA+ sera. Brief (20 min) exposure of EC to LA+ sera increased TF co-catalysis of Factor VII cleavage of Factor X (measured by chromogenic Xa substrate, S-2222) by up to 10 fold (p<0.05, unpaired t test). This effect was not the result of EC disruption or changes in whole-cell TF content. These data suggest multiple, complex and heterogenous effects of LA, including impaired production of PG12, impaired EC modulation, and heightened ability of endogenous EC tissue factor to initiate coagulation. These (and perhaps other) membrane-dependent effects may contribute to the tendency of LA+ patients to develop clots.

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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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In vivo studies of the role of factor VII in hemostasis

Blood ◽

10.1182/blood.v65.5.1197.bloodjournal6551197 ◽

1985 ◽

Vol 65 (5) ◽

pp. 1197-1200

Author(s):

AR Giles ◽

S Tinlin ◽

L Brosseau ◽

H Hoogendoorn

Keyword(s):

Alternative Pathway ◽

Factor Xa ◽

In Vivo Studies ◽

Factor Vii ◽

High Dose ◽

Factor X ◽

Clotting Factors ◽

Dose Warfarin ◽

Factor X Activation

The effect of both congenital and acquired factor VII deficiency on the cuticle bleeding time (CBT) was evaluated in dogs. The CBT has been previously documented to be a sensitive indicator of factor VIII:C deficiency in hemophilic dogs. Serial CBT determinations were made on normal dogs treated with high-dose warfarin. At 48 hours post- treatment, the CBT was normal, although the factor VII level was less than 1%, whereas the levels of factors II, IX, and X were 44%, 25%, and 17%, respectively. At 120 hours the CBT became abnormal when all vitamin K-dependent clotting factors had dropped to less than 18%. Administration of a plasma concentrate of factors II, IX, and X corrected the CBT, despite the factor VII level remaining at less than 1%. Similar studies in a congenitally factor VII-deficient dog (factor VII less than 2%) confirmed that this deficiency state was not associated with an abnormality of the CBT. Administration of heparin to both normal and factor VII-deficient animals was associated with prolongation of the CBT, but the heparin dose required in the normal animals was substantially higher than in the factor VII-deficient animals. These data do not suggest that factor VII/VIIa has an exclusive role in generating factor Xa, either directly or indirectly, by way of factor IXa generation, in vivo. However, the increase in heparin sensitivity of the factor VII-deficient animals does suggest that factor VII/VIIa may, in some circumstances, present a significant alternative pathway of factor X activation, although the activation pathway involved cannot be determined from the studies performed.

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Initiation of the extrinsic pathway of coagulation by human and rabbit alveolar macrophages: a kinetic study

Blood ◽

10.1182/blood.v74.5.1583.bloodjournal7451583 ◽

1989 ◽

Vol 74 (5) ◽

pp. 1583-1590 ◽

Cited By ~ 1

Author(s):

MP McGee ◽

R Wallin ◽

FB Wheeler ◽

H Rothberger

Keyword(s):

Alveolar Macrophage ◽

Alveolar Macrophages ◽

Plasma Concentrations ◽

Factor Xa ◽

Factor Vii ◽

Factor X ◽

Extrinsic Pathway ◽

Activation Rate ◽

Factor X Activation

We examined assembly and expression of the factor X activating complex on human and rabbit alveolar macrophages. Kinetic parameters of the factor X activating reaction were determined by functional titrations of factors VII and X with macrophage tissue factor (TF) added. We found rapid activation of factor X to Xa on alveolar macrophage surfaces. Detection of rapid factor Xa formation on macrophages required addition of exogenous factors VII and X. At plasma concentrations of the purified factors, factor Xa was formed on freshly isolated macrophages at approximately 5.4 pmol/min/10(6) cells. After macrophage maturation in culture for 20 hours with LPS (endotoxin) added, the factor X activation rate was increased two- to sixfold. The km' (apparent km) of TF-factor VII enzymatic complexes assembled on alveolar macrophages for factor X were (258 +/- 55 and 475 +/- 264 nmol/L for human and rabbit cells, respectively). The km' did not change during macrophage maturation in culture, but V'max (apparent Vmax) was consistently increased. The K1/2 of human factor VII (concentrations giving half maximal rates of factor X activation) for the interaction with human and rabbit alveolar macrophage TF were 0.191 +/- 0.096 and 1.7 +/- 0.7 etamol/L, respectively. The K1/2 were not significantly changed after maturation, whereas rates of Xa formation at saturation with factor VII were increased. The fast rates of factor X activation observed at physiologic concentrations of plasma-derived factors VII and X indicate that TF on alveolar macrophages is likely to provide sites for binding of factor VII and activation of factor X in vivo during clotting reactions associated with alveolar edema and inflammation.

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