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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1319-1326.)
© 1996 American Heart Association, Inc.

Articles

Hypercoagulable State in Patients With Antiphospholipid Syndrome Is Related to High Induced Tissue Factor Expression on Monocytes and to Low Free Protein S

Joan-Carles Reverter; Dolors Tassies; Josep Font; Joan Monteagudo; Gines Escolar; Miguel Ingelmo; Antoni Ordinas

the Servicio de Hemoterapia y Hemostasia (J.-C.R., D.T., J.M., G.E., A.O.) and the Unidad de Enfermedades Autoinmunes Sistemicas (J.F., M.I.), Hospital Clinic i Provincial, Barcelona, Spain.

Correspondence to Dr J.C. Reverter, Servicio de Hemoterapia y Hemostasia, Hospital Clinic, Villarroel 170, Barcelona 08036, Spain.

	Abstract

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Antiphospholipid antibodies (aPLs) are associated with thrombosis, but the mechanisms of this thrombotic tendency are unknown. We studied 56 patients (12 with systemic lupus erythematosus [SLE] and aPLs and previous thrombosis, 12 with SLE and aPLs but no thrombosis, 15 with SLE without aPLs or thrombosis, 11 with primary antiphospholipid syndrome with thrombosis, and 6 asymptomatic subjects with aPLs) to investigate the ability of aPLs to induce tissue factor (TF) expression on human normal monocytes. A double direct immunofluorescence technique (anti-CD14 and anti-TF) was used, and procoagulant activity in viable and disrupted cells was measured after plasma incubation for 6 hours at 37°C with normal mononuclear cells. Hemostasis regulatory proteins, prothrombin fragment 1+2, and thrombin–antithrombin III complex levels were determined. Increased TF expression and procoagulant activity were observed using plasma samples from SLE patients with aPLs and thrombosis (P<.01) and from primary antiphospholipid syndrome patients (P<.01) but not from patients with SLE and aPLs but no thrombosis, patients with SLE without aPLs, or asymptomatic patients with aPLs. Purified aPL immunoglobulins from one primary antiphospholipid syndrome and two SLE patients added to normal plasma showed a significant increase in both TF expression and procoagulant activity (P<.05) compared with purified aPL from two SLE patients without thrombosis. The addition of nonspecific IgG from three SLE patients without aPLs and from three control subjects did not increase TF expression. Low free protein S was seen in eight patients. Increased TF expression and low free protein S correlated with thrombosis (P<.01) and with higher prothrombin fragment 1+2 and thrombin–antithrombin III values (P<.01). These observations may contribute to a further understanding of the thrombotic risk in aPL patients.

Key Words: antiphospholipid syndrome • tissue factor • monocytes • free protein S • thrombosis

	Introduction

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The wide group of autoantibodies directed against negatively charged phospholipids includes aCLs and LA.¹ aPLs have been associated with an increased risk of arterial or venous thrombosis, recurrent pregnancy loss, and/or thrombocytopenia in patients with SLE² or PAS.^{1 3} Despite the association between aPLs and thrombosis in these patients, the mechanisms of the thrombotic tendency and the role of aPLs in the pathogenesis of thrombosis remain unknown.

Several mechanisms have been proposed to explain the development of thrombotic events, including inhibition of the release of prostacyclin by the endothelium,^{4 5} impairment of fibrinolysis,^{6 7 8} alterations in proteins C and S,^{9 10 11 12 13 14 15} and a direct procoagulant effect on platelets.^{16 17} The presence of aPLs may predispose subjects to thrombosis by inducing procoagulant TF activity on endothelial cells and monocytes.^{18 19 20 21 22 23} TF, an integral membrane protein that constitutes the major physiological initiator of blood coagulation in vivo,^{24 25} is present on the surface of a variety of nonvascular cells and is normally not expressed by intravascular cells.^{24 25} TF can be induced in monocytes and endothelial cells by a number of different stimuli,^{26 27 28 29} and aPLs could affect hemostasis through this mechanism.

The present study was performed to study TF expression and PCA induced on normal monocytes by plasma from patients with aPLs and the relationship of aPLs with coagulation regulatory proteins and clinical manifestations.

	Methods

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Patients
This study, which was approved by the Human Experimental Committee of the Hospital Clinic, was performed according to the principles of the Declaration of Helsinki. Informed consent was obtained from all participants.

Fifty-six individuals were included in this study: 39 patients fulfilled the revised criteria of the American Rheumatism Association for the diagnosis of SLE,³⁰ 11 patients had been diagnosed with PAS on the basis of clinical and laboratory data³ (9 women and 2 men, aged 32.6±7.0 years), and 6 individuals (5 women and 1 man, aged 31.5±10.3 years) had aPLs detected in laboratory tests in at least two determinations performed 3 months apart but no clinical manifestations (Table 1). Of the 39 SLE patients, 12 had aPLs and previous thrombosis (12 women, aged 38.8±13.6 years), 12 had aPLs but no previous thrombosis (11 women and 1 man, aged 36.6±8.4 years), and 15 had neither detectable aPLs nor a history of thrombosis (14 women and 1 man, aged 39.9±14.2 years). All PAS patients had a history of thrombotic events. All thrombotic events were assessed clinically and confirmed by venogram or arteriography. Time between thrombosis and blood sampling was at least 6 months (range, 6 to 55 months). All patients were followed up for at least 2 years (range, 2 to 12 years; median, 5.2 years). Median follow-up in the thrombosis group was 5.5 years and in the no-thrombosis group, 5.0 years. Plasma samples from 40 healthy individuals without autoimmune disease, bleeding disorders, thrombosis, or pregnancy loss were used as controls. None of the patients or control subjects had either thrombocytopenia or a positive Coombs test at the time the study was performed. None had been treated with aspirin, oral anticoagulants, or corticosteroids in the previous 2 weeks.

View this table: [in this window] [in a new window]   Table 1. Patient Characteristics

With the patient resting in a seated position, venous blood samples were drawn from a clean antecubital venipuncture without venocclusion and placed in tubes containing 3.8% trisodium citrate (1:9, vol/vol) (Becton Dickinson). Platelet-free plasma was obtained by a double centrifugation (2000g for 10 minutes at 22°C and 5000g for 10 minutes at 4°C). Plasma was divided into aliquots, snap-frozen in a mixture of dry ice and ethanol (1:2, vol/vol), and stored at -70°C.

Detection of LA
Prothrombin time, activated partial thromboplastin time, diluted Russell's viper venom time, and phospholipid inhibition were assessed by following the guidelines of the Subcommittee for the Standardization of Lupus Anticoagulants of the International Society of Thrombosis and Hemostasis.³¹ To rule out any defect in a coagulation factor, tests were performed with mixtures of patient and control subject plasmas.

Detection of aCLs
aCLs were measured in all patients by using a standardized ELISA.³² Results were expressed as binding index (BI) with respect to OA values of normal pooled serum as follows: Values were considered positive when the logarithms of the binding indexes were >98th percentile of the cumulative distribution of the control group. A semiquantitative value was given for aCLs according to a progressive scale³² (Table 1).

Immunoglobulin Purification
Sera from five patients with aCLs were obtained in sufficient quantity to perform affinity immunoglobulin purification by incubation with cardiolipin liposomes.³³ These patients had SLE with aPLs and thrombosis (Nos. 5 and 9), SLE and aPLs without thrombosis (Nos. 14 and 21), and PAS (No. 33). aCL activity was checked in the purified immunoglobulins by using an ELISA. In addition, nonspecific IgG subclass immunoglobulins were purified by using protein G–Sepharose columns (Pharmacia) from the sera of three aPL-negative SLE patients and three control subjects.

Hemostasis Studies
Antithrombin III activity was measured by using Behrichrom Antithrombin III (Behringwerke).³⁴ Protein C activity was quantified by using a colorimetric assay (Chromogenix).³⁵ Protein S and free protein S were evaluated by using ELISA techniques (Asserachrom Protein S and Asserachrom Free Protein S, Stago).³⁶ Plasminogen was evaluated by using a chromogenic assay (Chromogenix).³⁷ Plasma levels of F₁₊₂³⁸ and TAT complexes³⁹ were assessed by using ELISA techniques (Enzygnost-F₁₊₂ and Enzygnost-TAT, Behringwerke).

Monocyte Isolation and Induction of Monocyte TF Expression
Blood anticoagulated with citrate-phosphate-dextrose (final concentration of citrate in blood, 19 mmol/L) was obtained from normal volunteers (blood group O) by clean venipuncture. MBCs were isolated by sedimentation on a Ficoll-Paque (Pharmacia) gradient. After washing in PBS, pH 7.2 (Bio-Merieux), MBCs were immediately resuspended in undiluted plasmas from patients or control subjects and adjusted to a concentration of 20x10⁶ cells/mL. As determined by using the Trypan blue exclusion technique, cell viability in all preparations was always >96%. Percentages of monocytes as determined by cytochemical reactivity for -naphthyl butyrate esterase staining and flow cytometry using the phycoerythrin-tagged monoclonal antibody CD14 (LeuM3, Becton-Dickinson) ranged from 24% to 31% (26.8±1.5%). MBCs were incubated with patient or control subject plasmas for 6 hours at 37°C. This period of time was selected because in others'²⁶ and our own preliminary experiments 6 hours yields the highest effect on monocyte TF expression. For negative controls, MBCs were incubated with normal plasmas, and for positive controls, they were stimulated with 10 µg/mL LPS (Difco). MBCs were processed immediately after incubation. Several experiments were performed with affinity-purified aCLs or protein G–purified nonspecific IgG. In these experiments immunoglobulins were added to normal plasmas at a concentration of 50 µg/mL before being incubated with MBCs. Plasmas from control subjects and patients and supernatants of incubated MBCs were tested for endotoxin by using a test based on the activation of an enzyme system in the limulus amoebocyte lysate (Kabi Diagnostica). In all cases the amount of endotoxin was below the test detection limit.

Monocyte TF Expression by Flow Cytometry
Surface expression of TF on monocytes was measured by using a direct double-color immunofluorescence technique. After incubation with plasmas, MBCs were placed in separate tubes and washed with PBS, pH 7.2, containing 1% bovine serum albumin (Sigma) plus 1% human serum (blood group AB). Cells were incubated with the phycoerythrin-tagged monoclonal antibody anti-CD14 and the fluorescein-tagged monoclonal antibody anti-TF (American Diagnostica) for 30 minutes at 22°C. Nonspecific binding was assessed by using an irrelevant isotype-matched monoclonal antibody. Flow cytometric analysis was performed on a fluorescence-activated cell sorter (Becton Dickinson) by using Lysys II software.

Monocytes were monitored by using forward scatter (a measure of cell size related to cell cross section and refractive index), side scatter (related to the internal structure or granularity of the particle), and FL2 gates (fluorescence at 585 nm, phycoerythrin) and read in FL1 (fluorescence at 530 nm, fluorescein). Fluorescence data of 5x10³ monocytes were collected by using logarithmic amplification. Quadrant analysis of two-color dot-plot histograms was performed by setting the horizontal and vertical cursors on the isotypic negative control population to ensure there were <0.5% positive cells, and the percentage of TF-positive monocytes and the MFI, expressed in arbitrary fluorescence units at logarithmic settings, were recorded. Plasmas from patients that induced an MFI >mean+3xSD of control subject plasmas were considered positive for increased induced TF expression.

Determination of PCA
PCA was determined in triplicate by using a single-stage clotting assay on the surface of viable cells and in cell lysates. After incubation with plasmas, MBCs were washed with PBS, pH 7.2, resuspended (10⁶ monocytes/mL) in PBS, and used in the clotting assay. In the assay, 100 µL of the viable MBC suspension, 100 µL of cell lysates obtained by three cycles of freezing and thawing, or 100 µL of thromboplastin standards were incubated with human platelet-free citrated plasma (pooled from 20 normal donors) for 1 minute at 37°C. Coagulation was started by the addition of 100 µL of 25 mmol/L CaCl₂, and clotting times were recorded in a KC10 system (Amelung). PCA was determined by reference to a standard curve generated with serial dilutions of a commercial rabbit brain thromboplastin (Dade). A value of 10⁵ mU was arbitrarily assigned to a 40 mg (dry wt)/mL concentration of the standard thromboplastin. A standard log-log curve was calculated by using the serial dilutions of thromboplastin (Pearson's r always >0.98), from which PCA was determined for the test samples. Results were expressed as milliunits per 10⁵ monocytes. The assay was linear between 1 to 10⁵ mU. PCA was reduced by 88.1% by incubation of the viable cells or lysates for 2 hours with 50 µg/mL of a blocking anti-TF monoclonal antibody (American Diagnostica).

Statistical Analysis
Results are expressed as mean±SD. ANOVA, t test, ² test (with Yates' correction when appropriate), or Fisher's exact test were used for comparisons. Linear correlations were calculated by using the least-squares method. A value of P<.05 was significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Patient Characteristics
Table 1 describes clinical data of the 41 subjects with aPLs included in the study. LA was present in 27 patients, IgG subclass aCLs were found in 33, and IgM subclass aCLs in 29. Four of the PAS patients had previous fetal losses (No. 25 with one episode in the 26th week, No. 27 with two episodes in the 21st and 24th weeks, No. 29 with one episode in the 22nd week, and No. 35 with two episodes in the 10th and 31st weeks). One patient (No. 28) had an episode of autoimmune hemolytic anemia 4 years before. None of the patients had a history of autoimmune thrombocytopenia or leukopenia.

Hemostasis Studies
Low protein C values (<60%) were observed in three patients: No. 18, with SLE and aPLs but no previous thrombosis (55%), and in two PAS patients (No. 28 [52%] and No. 35 [58%]).

Normal total protein S values were seen in all the individuals (97.6±18.4%). Free protein S values lower than the normal threshold (60%) were seen in eight patients: three with SLE, aPLs, and thrombosis (No. 9 [51%], No. 10 [46%], and No. 12 [44%]), four with PAS (No. 29 [47%], No. 32 [57%], No. 33 [44%], and No. 34 [42%]), and one with SLE but no aPLs (No. 45, 51%). Low free protein S values were significantly associated with previous thrombotic events (7 of 23 patients with thrombosis versus 1 of 33 patients without thrombosis, P<.01).

Antithrombin III and plasminogen values were within normal range in all the patients (105.3±16.9% and 102.9±14.9%, respectively).

TF Expression on Monocytes
Basal TF expression on monocytes was low (MFI, 8.9±4.1 arbitrary fluorescence units; TF-positive monocytes, 8.8±3.5%). In control experiments, MBC incubation with normal plasmas did not show a significant increase in TF expression, whereas MBC incubation with normal plasmas plus LPS showed a significant (P<.01) increase (Table 2). Figs 1 and 2 and Table 2 show the results obtained after MBC incubation with patient plasmas. Increased expression of TF on normal monocytes was observed after incubations with plasmas from SLE patients with aPLs and thrombosis (P<.01). No increase in monocyte TF expression was observed after incubation of MBCs with plasmas from SLE patients who had neither aPLs or thrombosis or from SLE patients with aPLs but no thrombosis. The addition of plasma from PAS patients resulted in a clear increase of TF expression (P<.01), but plasmas from aPL-positive individuals without clinical manifestations had no apparent effect on the parameters evaluated.

View this table: [in this window] [in a new window]   Table 2. Induced TF Expression on Monocytes and PCA

View larger version (22K): [in this window] [in a new window]   Figure 1. Bar graph shows percentages of monocytes expressing TF as assessed by flow cytometry (solid bars) and PCA as determined by one-stage clotting assay in viable cells (hatched bars) or lysates (open bars). Differences observed in TF and PCA when MBCs were incubated with plasmas from SLE patients with aPLs and thrombosis (SLE aPL+ T+; n=12) or PAS (n=11) were significant (*P<.01) vs control subjects (n=40). No significant differences were observed when plasmas from asymptomatic individuals with aPLs (A-aPL; n=6), SLE patients without aPLs or thrombosis (SLE aPL-; n=15), or SLE patients with aPLs but without thrombosis (SLE aPL+ T-; n=12) were used. Values are mean±SD.

View larger version (19K): [in this window] [in a new window]   Figure 2. Flow cytometry histograms of TF expression on normal monocytes after incubation for 6 hours at 37°C with plasmas from control subjects and patients (A) No. 1, an SLE patient with aPLs and thrombosis (SLE APL+), (B) No. 25, a PAS patient, and (C) No. 36, an asymptomatic subject with aPLs (ASYMPTOMATIC APL+).

Twenty plasma samples showed increased induced TF expression on monocytes. Seven of these samples were from patients with SLE, aPLs, and thrombosis; two were from patients with SLE and aPLs but no thrombosis; two were from patients with SLE but no aPLs; and nine were from PAS patients. Increased induced TF expression was associated with a history of thrombosis (P<.01).

PCA in Viable and Disrupted Monocytes
Basal PCA was 6.7±4.2 mU/10⁵ monocytes using viable monocytes and 20.2±6.8 mU/10⁵ monocytes using lysates. MBC incubation with control plasmas did not show a significant increase in PCA (Table 2). However, MBC incubation with normal plasmas plus 10 µg/mL LPS showed a significant increase (P<.01) in PCA in both viable cells and lysates (Table 2). Increased PCA was observed by using plasmas from SLE patients with aPLs and thrombosis (P<.01) and from PAS patients (P<.01) but not by using plasmas from patients with SLE but no aPLs, with SLE and aPLs but no thrombosis, or from aPL-positive asymptomatic individuals (Fig 1 and Table 2).

A linear correlation was observed between PCA in viable cells and surface TF expression on monocytes as measured by flow cytometry (r=.64 for TF expression measured as MFI and r=.58 for TF expression measured as TF-positive monocytes, both P<.01).

Thrombin Generation
Plasma levels of F₁₊₂ and TAT are shown in Table 3. Patients with increased induced TF expression had higher F₁₊₂ (1.8±0.8 versus 0.8±0.4 nmol/L, P<.01) and TAT (13.2±16.2 versus 4.2±5.3 ng/mL, P<.01) values than patients with normal induced TF expression. In addition, patients with low values of free protein S had significantly higher F₁₊₂ (2.0±1.0 versus 1.0±0.5 nmol/L, P<.01) and TAT (16.1±21.6 versus 6.0±8.1 ng/mL, P<.05) values than patients with normal free protein S levels. F₁₊₂ values were also increased in patients with a history of thrombosis (1.4±0.7 versus 0.9±0.6 nmol/L, P<.01).

View this table: [in this window] [in a new window]   Table 3. F1+2 and TAT Complexes

Experiments With Purified Immunoglobulins
A significant increase was seen in both monocyte-induced TF expression (P<.05) and PCA (P<.05 in both viable cells and lysates) in plasma samples from the three patients with previous thrombosis (two with SLE and one with PAS) compared with the two SLE patients with aPLs but no thrombosis (Table 4). We observed no differences with the use of nonspecific IgG subclass immunoglobulins obtained from SLE patients without aPLs or thrombosis compared with nonspecific IgG from control subjects.

View this table: [in this window] [in a new window]   Table 4. Induced TF Expression on Monocytes and PCA After Incubation With Normal Plasma Plus Purified Immunoglobulins

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results of the present study demonstrate that plasmas from patients with aPLs and previous thrombosis can induce increased TF expression on normal monocytes and produce increased PCA. This may be one of the mechanisms that contributes to the pathophysiology of thrombosis associated with aPLs.

The mechanisms of thrombosis in patients with aPLs are a matter of controversy. Early reports suggested that aPLs led to thrombosis by causing impaired vascular prostacyclin production as a result of aPL binding to the phospholipids present on endothelial cells.^{4 5} However, only a few aPLs can bind to endothelial cells, and those that do bind do not necessarily demonstrate antiprostacyclin activity.^{40 41} Platelet activity^{5 16 17 42 43} and several other mechanisms involving blood coagulation and fibrinolysis^{6 7 8 9 10 11 12 13 14 15} have also been proposed in the thrombogenesis of aPL-positive patients. The inhibitory effect of aPLs on protein C activation has been observed in endothelial cells in vitro.^{9 10 11} In addition, in aPL-positive patients inhibition of fibrinolysis^{6 7 8} has been proposed. Other authors^{13 14} have suggested deficiencies of protein S as a pathophysiological mechanism of thrombosis in these patients. In the present study, low values of free protein S were seen in a significant number of patients with thrombosis together with increased levels of thrombin generation markers (F₁₊₂ and TAT). However, total protein S levels were in the normal range in all cases. The discrepancy between total and free protein S in SLE patients has been attributed to an increase in C4b-BP, an acute-phase reactant, or to a change in C4b-BP affinity to protein S.¹² Patients with this (presumably acquired) alteration in the hemostasis regulatory protein S may not be able to compensate for the procoagulant tendency due to the action of aPLs in platelets, endothelial cells, or monocytes, in the same way that has been proposed as an explanation for the activated protein C resistance seen in these patients.⁴⁴

The complex interactions between cells and plasma coagulation proteins have gained importance in recent years as the explanation of the pathophysiology of thrombosis. TF is widely accepted to be the in vivo most important physiological initiator of blood coagulation, with clear implications for the development of thrombotic events.^{24 25} TF is normally not expressed by intravascular cells but can be induced in monocytes and endothelial cells by different physiological or nonphysiological stimuli, such as bacterial LPS, tumor necrosis factor, interleukin-1, or immune complexes.^{26 27 28 29} Both PCA and TF expression on monocytes are rapid events, reaching their maximum after 6 hours of stimulation²⁶ as result of a transient (de novo) transcription of the TF gene.⁴⁵

An increased PCA attributed to TF expression on monocytes induced by murine monoclonal aCLs that can induce antiphospholipid syndrome has been found in mice,²¹ but this may or may not be applicable to humans. The induction of endothelial PCA by sera or immunoglobulins from patients with antiphospholipid syndrome has been reported,^{18 19 20 23} but increased PCA has been reported in SLE patients with or without the presence of LA.²² In the present study we compared the ability of plasmas to promote monocyte TF expression (as detected by flow cytometry) and TF-dependent PCA from aPL-positive patients (SLE patients with and without previous thrombotic events and PAS patients), from SLE patients without thrombosis or aPLs, and from asymptomatic individuals with aPLs. Our results indicate that only those plasmas from patients with aPLs and previous thrombosis increased monocyte TF expression after incubation with normal monocytes. These results are consistent with the measured PCA, which showed a relatively good correlation with TF expression, a determination that is not influenced by PCA inhibitors.²⁶ Purified immunoglobulins showed results similar to those obtained with plasmas that showed increased TF expression and PCA only from patients with aPLs and previous thrombotic events. In addition, patients with high monocyte TF expression showed increased levels of plasma TAT and F₁₊₂, suggesting increased thrombin generation. This high induced TF may reflect or even cause the hypercoagulable state. Our findings support the hypothesis that TF expression by monocytes may play an important pathogenic role in thrombosis in both the arterial and venous circulation. Monocytes, which are circulating blood elements with a key role in the inflammatory response, provide an appropriate membrane site for the assembly of coagulation proteases. We believe that increased expression of TF by monocytes in SLE patients with aPLs and thrombosis and in PAS patients may contribute to the activation of the coagulation mechanisms that led to many of the thrombotic events.

The fact that plasmas from patients with aPLs promoted monocyte TF expression only in cases with a previous history of thrombosis but not in aPL-positive asymptomatic individuals or nonthrombotic SLE patients provides indirect evidence for the heterogeneity of aPLs. The existence of subgroups in aPLs^{1 3 15} has been reported, and the different clinical findings could be influenced by the fact that antibodies may recognize different target antigens. A different effect of aPLs on PCA has been described according to the charge of the phospholipids against which they are directed.⁴⁶ In this way, some aPLs do not recognize phospholipids alone but can recognize ß₂-glycoprotein I or a phospholipid–ß₂-glycoprotein I complex.^{47 48} Those aPLs that can recognize ß₂-glycoprotein I may have a greater potential to be involved in the pathogenesis of thrombosis.⁴⁹

The mechanism by which aPLs induce TF-dependent PCA on endothelial cells or monocytes is unknown.¹⁹ However, a linkage between aPLs and TF expression can be suggested. TF requires interaction with membrane phospholipids to become functionally active,⁵⁰ and TF activity is enhanced by phosphatidylserine.^{50 51} Since aPLs bind to negatively charged phospholipids such as phosphatidylserine,¹ it is possible that aPLs may enhance TF expression through interaction with adjacent phospholipids.¹⁹ In any case, the delayed induction of TF expression, which needs several hours to be developed, strongly suggests the requirement of TF synthesis by the activated cells. Furthermore, TF expressed on the surface of stimulated monocytes may play an additional role through promotion of platelet thrombus formation.⁵²

Interaction between TF-positive monocytes and platelets may be mediated by a small local thrombin formation that is factor VIIa dependent and able to activate platelets; subsequently, tenase and prothrombinase complexes assemble on the activated platelet surface, thus generating the final thrombus.⁵³ Such a mechanism would be consistent both with our previous findings,^{16 17} which show increased platelet thrombi formation in normal citrate-anticoagulated blood perfused through a collagen-rich chamber (denuded rabbit aorta) after incubation with aPL-positive plasmas from patients with a history of thrombosis, and with the increased induced TF expression and elevated thrombin generation markers observed in the present series of patients.

	Selected Abbreviations and Acronyms

 

aCL = anticardiolipin antibody aPL = antiphospholipid antibody ELISA = enzyme-linked immunosorbent assay F1+2 = prothrombin fragment 1+2 LA = lupus anticoagulant LPS = lipopolysaccharide MBC = mononucleated blood cell MFI = mean channel of fluorescence intensity OA = optical absorbance PAS = primary antiphospholipid syndrome PBS = phosphate-buffered saline PCA = procoagulant activity SLE = systemic lupus erythematosus TAT = thrombin–antithrombin III TF = tissue factor

	Acknowledgments

 
This work was supported by Spanish government grants (FIS 92/0782, FIS 94/5402, FIS 95/0250, and FIS 96/0580) from the Fondo de Investigaciones Sanitarias.

Received December 5, 1995; revision received July 16, 1996;

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