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Published ahead of print on November 15, 2007, doi:10.1164/rccm.200706-840OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 536-543, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200706-840OC

Original Article

Procoagulant Membrane Microparticles Correlate with the Severity of Pulmonary Arterial Hypertension

Babe Bakouboula1,2,3,*, Olivier Morel1,2,3,*, Antoine Faure1, Fatiha Zobairi2,3, Laurence Jesel1, Annie Trinh1, Michel Zupan1, Matthieu Canuet4, Lelia Grunebaum5, Agnès Brunette6,7, Dominique Desprez5, François Chabot6,7,8, Emmanuel Weitzenblum4, Jean-Marie Freyssinet2,3, Ari Chaouat4,8,** and Florence Toti2,3,9,**

1 Hôpitaux Universitaires de Strasbourg, Fédération de Cardiologie, Strasbourg, France; 2 Université Louis Pasteur, Institut d'Hématologie et d'Immunologie, Strasbourg, France; 3 INSERM, U.770, Le Kremlin-Bicêtre, France; 4 Hôpitaux Universitaires de Strasbourg, Département de Pneumologie, Strasbourg, France; 5 Hôpitaux Universitaires de Strasbourg, Hématologie Biologique, Service d'Hémostase, Strasbourg, France; 6 Faculté de Médecine de Nancy, Nancy Université, Vandoeuvre-lès-Nancy, France; 7 INSERM, U.734, Faculté de Médecine de Nancy, Vandoeuvre-lès-Nancy, France; 8 Service des Maladies Respiratoires et Réanimation Respiratoire, Centre Hospitalier Universitaire de Nancy, Vandoeuvre-lès-Nancy, France; and 9 Faculté de Médecine, Université Paris-Sud 11, Le Kremlin-Bicêtre, France

Correspondence and requests for reprints should be addressed to Pr. Ari Chaouat, M.D., Service des Maladies Respiratoires et Réanimation Respiratoire, Hôpital d'adultes de Brabois, Allée du Morvan, 54511 Vandoeuvre-lès-Nancy Cedex, France. E-mail: a.chaouat{at}chu-nancy.fr


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Procoagulant microparticles constitute valuable hallmarks of cell damage. Microparticles also behave as cellular effectors.

Objectives: We hypothesized that the extent of the vascular cell damage measured by circulating microparticles could be related to the severity of pulmonary arterial hypertension (PAH).

Methods: Circulating biomarkers of vascular damage and cell activation were measured in blood samples from 20 patients with PAH. Samples were withdrawn from occluded pulmonary artery and jugular vein. Peripheral venous blood samples were obtained in 23 control subjects. The microparticle procoagulant abilities were quantified by functional prothrombinase and tissue factor assays and their cellular origin was determined.

Measurements and Main Results: Soluble vascular cellular adhesion molecule-1 and proinflammatory markers, such as monocyte chemoattractant protein-1 and highly specific C-reactive protein, were elevated in patients with PAH compared with control subjects. Microparticles bearing active tissue factor and CD105 (endoglin) were also elevated in patients with PAH compared with control subjects (29 ± 13 vs. 16 ± 6 fmol/L, P < 0.001, and 1.10 ± 0.46 vs. 0.49 ± 0.33 nmol/L phosphatidylserine equivalent, P < 0.001, respectively). A further increase in endothelium-derived CD105 microparticles was observed in pulmonary arterial blood compared with venous blood in patients with PAH (1.73 ± 0.77, P = 0.038). Microparticles bearing active tissue factor were at a higher level in patients in functional class III and IV and who were walking fewer than 380 m with the six-minute-walk test.

Conclusions: Circulating markers of endothelium damage, proinflammatory markers, and cell stimulation estimated with circulating microparticles appear to be valuable tools in determining the severity of PAH.

Key Words: pulmonary hypertension • endothelium • tissue factor • VCAM-1 • endoglin



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Procoagulant microparticles are circulating markers of thrombotic tendency in cardiovascular disorders. There is no information on their possible role in the pathophysiology of pulmonary arterial hypertension.

What This Study Adds to the Field
Procoagulant microparticles correlate with the severity of pulmonary arterial hypertension.

 
Pulmonary arterial hypertension (PAH) is a severe disease of the small pulmonary arteries characterized by vascular narrowing and raised pulmonary artery pressure leading to the development of right-sided heart failure and death (1). In PAH, vasoconstriction, remodeling of the pulmonary vessel wall, endothelial and vascular smooth muscle cell proliferation and dysfunction, and thrombosis contribute to increased pulmonary vascular resistance (PVR), right ventricle overload, and stretch. Uncontrolled endothelial cell proliferation leading to the formation of plexiform lesions is frequently described. In situ, both hemostatic and fibrinolytic functions of the endothelium are altered, as suggested by the elevated plasma levels in von Willebrand factor (vWF), P-selectin, and plasminogen activator inhibitor type-1 (PAI-1), and decreased thrombomodulin plasma concentrations (2). Thrombotic lesions and platelet dysfunction are common features in PAH. In the flowing blood, circulating endothelial cells exhibit CD36 and E-selectin, two markers that correlate with pulmonary hemodynamic parameters (3).

The up-regulation of several cytokines and their elevated plasma levels has been demonstrated in severe PAH, emphasizing a possible influence on inflammatory mechanisms (4). The importance of such inflammatory mechanisms is underlined by the correlation observed between inflammatory markers and PVR (5). Interactions between activated platelets and the endothelium could also lead to thrombus formation and to the release of bioactive effectors of pulmonary vasoconstriction and remodeling. As an example, CD40L, a transmembrane protein found on lymphocytes and activated platelets, would thus contribute to inflammation by promoting the up-regulation and release of IL-8 and monocyte chemoattractant protein (MCP)-1 chemokines, leading to lung perivascular invasion by macrophages and lymphocytes. Furthermore, inflammatory cell infiltrates have been detected in plexiform lesions of pulmonary hypertension.

In the vasculature, procoagulant microparticles shed by apoptotic or stimulated cells upon membrane blebbing constitute valuable hallmarks of cell damage (6–8). Elevated levels of platelet-, endothelial-, monocyte-derived microparticles are a common feature of most thrombotic diseases, including pulmonary embolism (9–12). Indeed, circulating microparticles provide an additional phospholipid surface for the assembly of clotting enzyme complexes promoting thrombin generation. Their catalytic property relies on a procoagulant anionic aminophospholipid, phosphatidylserine, translocated to the exoplasmic leaflet after membrane remodeling, and on the possible presence of tissue factor, the main cellular initiator of blood coagulation. Circulating microparticles also behave as cellular effectors and may contribute to vascular inflammation and endothelial dysfunction. In the target cell, microparticles may promote cytokine release, cytoadhesin up-regulation, nitric oxide synthase inhibition, reduction of nitric oxide bioavailability, or redox balance impairment (7).

We hypothesized that microparticle generation, endothelial damage, and neurohormonal stimulation could be related to the severity of PAH assessed by pulmonary hemodynamic parameters, the six-minute-walk test (6MWT), and the World Health Organization (WHO) functional classification. Preliminary results of this study have been previously reported in the form of an abstract (13).


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Control Subjects
We studied 20 patients with severe PAH (75% in WHO functional classes III and IV); 11 patients were recorded as having idiopathic PAH (IPAH), according to the criteria of the Third World Symposium on PAH (Venice, Italy, 2003). Other etiologies were PAH associated with connective tissue disease (n = 5), HIV infection (n = 2), and portal hypertension (n = 2). Twenty-three patients referring to the cardiology department for atypical chest pain with a normal cardiovascular profile were enrolled as control subjects. Infectious diseases, severe kidney failure, and acute coronary syndrome within the past 6 months were exclusion criteria. Individual predisposing factors for cardiovascular disease are listed in Table 1. In control subjects, no acute cardiovascular event was recorded in the previous year. Written, informed consent was obtained from all patients with approval of the local ethics committee (Alsace No. 1, France).


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  		TABLE 1. CHARACTERISTICS, TREATMENTS OF PATIENTS AND CONTROL SUBJECTS, AND RIGHT HEART CATHETERIZATION, AND ARTERIAL BLOOD GAS FINDINGS

 
Pulmonary Hemodynamic Studies
We used the Swan-Ganz standard technique for right heart catheterization. During right-sided heart catheterization, mean arterial pressure (mPAP) and pulmonary capillary wedge pressure (PCWP) were obtained. Cardiac output was determined by thermodilution. PVR was calculated. The diagnosis of PAH was confirmed when mPAP was superior to 25 mm Hg at rest with a normal PCWP (<15 mm Hg).

6MWT
The 6MWT was conducted in accordance with the American Thoracic Society guidelines (14).

Blood Sampling Protocols
During right-heart catheterization, blood samples were collected from the occluded pulmonary artery and the jugular vein. In the control group, blood samples were withdrawn by venipuncture of a forearm vein. Platelet-poor plasma (PPP) samples were obtained by double centrifugation as previously described (15).

Isolation of Circulating Microparticles, Determination of Their Procoagulant Potential
Procoagulant microparticles were captured from PPP onto insolubilized annexin-V and their phosphatidylserine content was measured by functional prothrombinase assay (15). Results were expressed as phosphatidylserine equivalent (PhtdSer Eq). We confirmed that the venous site, jugular vein versus forearm vein, did not account for differences of microparticle levels (Table E1 of the online supplement).

Search for the Cellular Origin of Circulating Microparticles
Biotinylated monoclonal antibodies to the following various cell types were insolubilized onto streptavidin-coated microtitration plates and incubated with PPP (15): (1) anti-CD11a for leukocytes; (2) anti-CD31, a dual probe for apoptotic endothelial cells and platelet stimulation; (3) anti–E selectin (CD62E) for stimulated endothelial cells (16, 17); (4) anti-CD105 (endoglin) and antifractalkine for endothelial cells; and (5) anti-glycoprotein Ib (GPIb) for platelets. Captured procoagulant microparticles were quantified by prothrombinase assay. In a given sample, no direct comparison between capture by annexin-V and by antibodies can be given because affinities for the respective ligands are different.

Assessment of Tissue Factor Activity Harbored by Microparticles
Microparticles bearing tissue factor (TF) were isolated by capture onto insolubilized biotinylated specific antibody to human TF and quantified using a standardized TF activity assay (Innovin; Dade Behring, Marburg, Germany). Values are expressed as fmol/L of active TF.

Measurement of Soluble P-Selectin, Soluble CD40L, Soluble Vascular Cellular Adhesion Molecule-1, Monocyte Chemoattractant Protein-1, IL-1β, and IL-6
Measurements were performed in PPP samples, as recommended by the manufacturer using the Flow Cytomix human cardiovascular multiplex fluorescent bead immunoassay (Bender MedSystems, Vienna, Austria).

Miscellaneous Measurements
RANTES (regulated upon activation, normal T-cell expressed and secreted) was quantified in PPP samples by ELISA. Quantitative determination of plasma vWF antigen (vWF:Ag) and highly specific C-reactive protein (hsCRP) was realized by immunoturbidimetric assays. PAI-1 plasma concentrations were determined using a synthetic chromogenic substrate method. Circulating brain natriuretic factor (BNP) levels were determined by enzyme immunoassays.

Statistical Analysis
Statistical analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL). Results are expressed as mean ± SD. P < 0.05 was considered significant.

Additional details of the methods used are provided in the online supplement.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients
Individual anthropometric data, treatments, and right-heart catheterization findings are given in Table 1. No significant difference in age, sex distribution, or pulmonary hemodynamic and arterial blood gas parameters could be demonstrated between patients with IPAH or associated PAH (data not shown). Control subjects were slightly younger, but the difference did not reach the significance level. Dyslipidemia was significantly more frequent in control subjects compared with patients with PAH.

Endothelium Damage in PAH
Higher levels of soluble vascular cellular adhesion molecule (sVCAM)-1 were measured in the jugular vein of patients with PAH, compared with values detected in venous blood of control subjects (1,977 ± 805 vs. 1,104 ± 670 ng/ml, P = 0.001, respectively). However, no difference was observed between mean levels of sVCAM-1 in jugular vein compared with occluded pulmonary artery in patients with PAH (Figure 1). PAI-1 concentrations were significantly higher in the plasma from patients with PAH compared with controls (19.8 ± 7.7 vs. 11.5 ± 6.7 U/ml; P < 0.01) and were inversely correlated with the extent of fibrinolysis assessed by D-dimers in patients with PAH (r = –0.805, P = 0.016). No significant difference could be evidenced between plasma levels of vWF:Ag measured in PAH and control subsets (1.66 ± 0.81 vs. 1.45 ± 0.38 U/ml, P = 0.7).


Figure 1
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  		Figure 1. Circulating levels of vascular cellular adhesion molecule (sVCAM)-1. sVCAM-1 was measured using a fluorescent bead immunoassay in the peripheral venous blood from control patients (CTL), and in the jugular vein (PAHJV) and the occluded pulmonary artery blood (PAHPA) from patients with pulmonary arterial hypertension (PAH). Values are expressed as ng/ml. ns = not significant.

 
Inflammatory Status in PAH
Higher concentrations of monocyte chemoattractant protein (MCP)-1 and RANTES proinflammatory chemokines, as well as hsCRP, were measured in the jugular vein of patients with PAH and compared with values obtained in the venous blood of control subjects (413 ± 237 pg/ml vs. 209 ± 228 mg/L, P < 0.01, and 1,584 ± 967 vs. 887 ± 634 mg/L, P = 0.01, respectively). Plasma levels of MCP-1 and RANTES measured in the jugular vein and occluded pulmonary artery of patients with PAH were comparable (Table 2). In occluded pulmonary artery blood, significant correlations between RANTES and CD31- or CD11a-bearing microparticles were shown (r = 0.552, P = 0.02, and r = 0.524, P = 0.03, respectively). MCP-1 levels were correlated with sVCAM-1 concentrations in both jugular vein and occluded pulmonary artery blood (r = 0.553, P = 0.02, and r = 0.572, P = 0.02, respectively). No statistical difference could be observed in IL-1β and IL-6 circulating levels between control subjects and patients with PAH (Table E2).


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  		TABLE 2. BIOLOGICAL CHARACTERISTICS IN PULMONARY ARTERIAL HYPERTENSION AND CONTROL SUBSETS

 
Procoagulant Microparticles in PAH
Patients with PAH compared with control subset.
Procoagulant microparticle levels measured by capture onto annexin-V were not significantly different in the jugular vein blood from patients with PAH (6.9 ± 4.4 nmol/L PhtdSer Eq) when compared with values measured in control subjects (5.4 ± 2.1 nmol/L PhtdSer Eq, P = 0.37) (Figure 2). Likewise, no difference in platelet- or leukocyte-derived microparticle levels was found (Table 2 and Table E2).


Figure 2
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  		Figure 2. Circulating procoagulant microparticles (MPs). MPs were isolated from the peripheral venous blood of control patients (CTL), and from the jugular vein (PAHJV) and the occluded pulmonary artery blood (PAHPA) from patients with pulmonary arterial hypertension (PAH). MPs were captured onto annexin-V and measured using functional prothrombinase assay. Values are expressed as nanomolars of phosphatidylserine equivalents (nM PhtdSer Eq). ns = not significant.

 
In patients with PAH, levels of endothelium-derived CD105-bearing microparticles (CD105+-MPs) were significantly increased when measured in the jugular vein compared with control subjects (1.10 ± 0.46 vs. 0.49 ± 0.33 nmol/L PhtdSer Eq, P < 0.001). Interestingly, a higher TF activity was harbored by microparticles (TF+-MPs) isolated from the jugular vein compared with control subsets (29.4 ± 13 vs. 15.7 ± 6 fmol/L, P < 0.001) (Table 2 and Figure 3).


Figure 3
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  		Figure 3. Circulating microparticles bearing active tissue factor (TF+-MPs). MPs were measured in the peripheral venous blood from control patients (CTL), and in the jugular vein (PAHJV) and the occluded pulmonary artery blood (PAHPA) from patients with pulmonary arterial hypertension (PAH). Procoagulant MPs were captured onto insolubilized biotinylated specific antibody to human TF, and TF-related procoagulant activity was measured as described in METHODS. Values are expressed as femtomoles (fM) per liter of active TF. ns = not significant.

 
Comparison between the two puncture sites in PAH subset.
In the PAH subset, endothelium-derived CD105+-MPs were detected at higher levels in occluded pulmonary artery compared with the jugular vein (Table 2 and Figure 4). Furthermore, in the jugular vein, levels of microparticles bearing active TF or exposing E-selectin, a marker of endothelial activation, were slightly correlated (r = 0.489, P = 0.039). Microparticle-borne TF activity in the pulmonary vascular bed was correlated to vWF:Ag concentration and BNP level assessed in the peripheral venous blood (r = 0.530, P = 0.029, and r = 0.664, P = 0.01, respectively). Notably, levels of procoagulant microparticles captured on annexin-V or CD105 antibody were higher in occluded pulmonary artery than in the jugular vein (Figures 2 and 4). All other categories of microparticles and soluble markers were similar in both puncture sites (Figures 1 and 3; Table 2 and Table E2). The above difference suggests a gradient of circulating microparticles across the precapillary lung circulation. Values of the microparticle gradient detected by capture on annexin-V were related to those obtained by capture on CD105 (r = 0.749, P = 0.008).


Figure 4
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  		Figure 4. Circulating procoagulant endothelium-derived microparticles bearing endoglin (CD105+-MPs) MPs were isolated from the peripheral venous blood from control patients (CTL), and from the jugular vein (PAHJV) and the occluded pulmonary artery blood (PAHPA) of patients with pulmonary arterial hypertension (PAH). MPs were captured onto anti-CD105 antibody and measured using functional prothrombinase assay. Values are expressed as nanomolars of phosphatidylserine equivalents (nM PhtdSer Eq).

 
Relationships between Clinical or Physiologic Parameters and Circulating Biomarkers
The PVR, a physiologic surrogate of distal pulmonary vascular remodeling, was significantly correlated with BNP, a biomarker of right-heart failure in patients with PAH (r = 0.73, P = 0.002). BNP was correlated with sVCAM-1 (r = 0.59, P = 0.03), a biomarker of the endothelium damage, and with cardiac output (r = –0.77, P < 0.01). We also observed significant correlations between PVR and sVCAM-1 in jugular vein and between PVR and sVCAM-1 measured in occluded pulmonary artery blood. In addition, mPAP, cardiac output, and right atrial pressure, and sVCAM-1 level in occluded pulmonary artery, were correlated (Table 3). Stepwise multiple linear regression analysis with PVR as a dependent variable demonstrated that BNP was the strongest independent predictor of increase in PVR.


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  		TABLE 3. CORRELATIONS BETWEEN sVCAM-1 IN JUGULAR VENOUS BLOOD AND IN OCCLUDED PULMONARY ARTERY BLOOD IN PATIENTS WITH PULMONARY ARTERIAL HYPERTENSION, PULMONARY HEMODYNAMIC FINDINGS AND BRAIN NATRIURETIC PEPTIDE LEVEL

 
It must also be mentioned that distance in the 6MWT (6MWD), the most frequently used primary endpoint in randomized trials dedicated to PAH, was negatively correlated with microparticles bearing active TF in jugular vein and occluded pulmonary artery blood (r = –0.52, P = 0.046, and r = –0.62, P = 0.017, respectively). This exercise test was also negatively correlated with BNP levels (r = –0.71, P = 0.003). Microparticles with TF activity were detected at higher levels in the subset of patients with PAH in WHO functional class 3 or greater, and with 6MWT results of less than 380 m (Figure 5). Stepwise multiple linear regression analysis with 6MWD as a dependent variable demonstrated that sVCAM-1 measured in the jugular vein was an independent predictor of decrease in exercise capacity.


Figure 5
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  		Figure 5. Relationships between circulating microparticles bearing active tissue factor (TF+-MPs) in the occluded pulmonary arterial blood (PAHPA) and functional status assessed by six-minute-walk test (A) and World Health Organization (WHO) functional classification (B). Patients with pulmonary arterial hypertension (PAH) were divided into two subsets according to the mean six-minute-walk distance (380 m) or WHO functional class (<3). Procoagulant microparticles were captured onto insolubilized biotinylated specific antibody to human TF, and TF-related activity was measured as described in METHODS. Values are expressed as femtomoles per liter of TF.

 
Values of the microparticle gradient observed across the lung precapillary circulation between the occluded pulmonary artery and the jugular vein was correlated with mPAP (r = 0.631, P = 0.01) (Figure 6).


Figure 6
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  		Figure 6. Correlations between the procoagulant microparticle (MP) gradient across the precapillary lung circulation and the mean pulmonary artery pressure (mPAP). Each MP gradient was calculated by subtracting the jugular venous blood level from the pulmonary artery level of MPs captured onto annexin-V.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that elevated levels of microparticles bearing active TF and endothelium-derived microparticles bearing CD105 (CD105+-MPs) circulate in the plasma from patients with PAH. In addition, we found that higher amounts of procoagulant microparticles, measured by capture on annexin-V, are present in the occluded pulmonary artery blood compared with jugular vein blood.

Other indicators of inflammation and vascular damage such as hsCRP, RANTES, MCP-1, and sVCAM-1 were found to be significantly elevated in patients with PAH compared with a control subset with normal cardiovascular background. Endothelial damage, neurohormonal activation, and circulating procoagulant microparticles appeared indicative of the severity of PAH assessed by hemodynamic or functional parameters.

Inflammatory Status in PAH
Previous reports emphasize a role for inflammation in PAH. Enhanced MCP-1 expression was reported in humans (18, 19) and anti–MCP-1 therapy inhibits the development of PAH in rats. Furthermore, an enhanced expression of RANTES, mainly originating from endothelial cells, was demonstrated in lung samples from patients with severe pulmonary hypertension (20). Such tissue expression was associated with inflammatory cell infiltration. In the present study, significant correlations between MCP-1 and sVCAM-1, a reliable probe of endothelial stimulation, were observed regardless of the puncture site, thereby confirming the presence of persisting vascular damage. In the pulmonary vascular bed, RANTES was associated with procoagulant microparticles of leukocyte (CD11a+-MPs), endothelial, and platelet origin (CD31+-MPs), possibly reflecting multiple pathways in which microparticles contribute to inflammation and thrombus formation.

Neurohormonal Activation
Previous studies in patients with PAH have demonstrated the role for BNP as a marker of increased right cardiac pressure and volume overload. BNP has been shown to correlate with hemodynamic parameters, functional status, and prognosis of patients with PAH (21–23). Our present study is in accordance with these reports. BNP was an independent predictor of PVR and was correlated negatively with cardiac output and 6MWD.

Endothelium Damage in PAH
In PAH, alterations of the pulmonary vascular circulation include smooth muscle cell proliferation and arteriolar muscularization, fibrosis, endothelial cell proliferation, intimal thickening, and in situ thrombosis. Endothelial cells may respond to injury in various ways, affecting the process of vascular remodeling, including proliferation, apoptosis, release of vasoactive agents, and growth factors (24). After inflammatory, hypoxic, or shear stress, endothelial cells display proadhesive, proinflammatory, and prothrombotic phenotypes and release sVCAM-1, vWF, PAI-1, and endothelium-derived microparticles. Our data strongly highlight the link between the severity of PAH and endothelial stimulation. An argument in support of this hypothesis is that sVCAM-1 concentrations were correlated to mPAP, PVR, cardiac output, and 6MWD. However, we could not establish whether the elevation in plasma endothelial markers is the result of a severe raise in intravascular pressures or rather constitutes a deleterious cell signal, possibly tuning the severity of the disease through leukocyte or platelet recruitment, altered fibrinolysis, or tissue proteolysis. In the present study, endothelial microparticle phenotypes may reflect a particular cellular response under the characteristic vascular settings of PAH (see below).

Procoagulant Microparticles in PAH: Noxious Effectors?
The combined alteration of coagulation and fibrinolysis was previously demonstrated in PAH using a variety of circulating markers, including tissue plasminogen activator, PAI-1, D-dimers, vWF, P-selectin, and thrombomodulin (2, 25–28). Altogether, a hemostatic imbalance resulting from increased thrombotic factors and diminished fibrinolytic activity is believed to favor thrombosis (29, 30).

Membrane microparticles constitute a reservoir of potent actors in the adjustment of the hemostatic balance (7). Because of their dual procoagulant abilities, endothelium-derived microparticles have been reported to be possible effectors in cardiovascular and thrombotic disorders (16, 31). Shed from the plasma membrane of damaged endothelial cells, they exhibit phosphatidylserine, eventually combined to TF, and can be endowed with proadhesive abilities (32, 33).

Active TF Borne by Microparticles and PAH Pathogenesis
Recently, a circulating reservoir of TF, mainly conveyed by microparticles, of various cell origins, has been termed "blood-borne tissue factor" (34, 35). Multiple fusions and exchanges between monocytes, endothelial cells, platelets, and derived microparticles would favor phosphatidylserine location at TF vicinity and promote the so-called deencryption of its active form (35–37). The role generally assigned to vessel-bound TF in the initiation of blood coagulation is now believed to require additional blood-borne TF in the swift growth of the thrombus (38, 39).

TF has been poorly investigated in PAH. In a rodent model of severe pulmonary hypertension, specific TF staining of plexiform-like lesions has been shown (40). Thrombin, a potent inducer of TF expression by pulmonary artery smooth muscle cells, would amplify TF-driven coagulation responses (41–43). In the present study, the elevated microparticle TF procoagulant activity detected in patients with PAH is indicative of vascular cell damage or stimulation. Furthermore, the highest TF+-MP levels were measured in patients with PAH with lesser functional capacity (6MWD < 380 m and WHO functional class >= 3). These results are in agreement with the hypothesis of a noxious prothrombotic effect of TF+-MPs in PAH.

Endoglin, a Possible Vascular Modulator at the Endothelial Microparticle Surface
In the present study, levels of procoagulant microparticles captured onto annexin-V did not reach statistical significance in the jugular vein. However, values of procoagulant microparticles measured in the occluded pulmonary artery from patients with PAH reached the high levels evidenced during acute cardiovascular diseases, such as unstable angina (44), myocardial infarction in non–diabetes mellitus patients (45), or acute cardiac rejection (unpublished data). Endothelial cells appeared to be a key contributor to procoagulant microparticle shedding because significant variations in sVCAM-1 as well as CD105-bearing microparticles could be demonstrated between PAH and control subsets. However, endothelium-derived microparticles expressing other phenotypes, such as CD31, E-selectin, or fractalkine, were not significantly elevated in the pulmonary microvasculature. These observations probably reflect the specific contribution of endoglin (CD105) in PAH and confirm the lower threshold of the other endothelial markers investigated, as observed by our laboratory in other pathologic settings. In PAH, the specific elevation in CD105-bearing microparticles emphasizes the importance of endoglin-mediated signalling in the pulmonary vasculature. Endoglin is an accessory receptor for transforming growth factor-β, which is involved in endothelial cell proliferation. In vivo, endoglin is predominantly expressed by angiogenetic vessels and by in vitro proliferating endothelial cells. Recently, it was demonstrated that the ectopic expression of endoglin promotes endothelial cell proliferation (46). In the present study, an eventual microparticle-mediated endoglin transfer to neighboring endothelial cells may occur and promote cell proliferation. Such intercellular transfer has been described for monocyte microparticles able to deliver CCR5 to endothelial cells or TF to activated platelets (37, 47).

A Gradient of Endothelial Circulating Microparticles across the Precapillary Lung Circulation
Interestingly, the characteristic procoagulant microparticle gradient measured between occluded pulmonary artery and jugular vein (by capture on annexin-V) was also evidenced for CD105-bearing microparticles. This observation is suggestive of the following: (1) an increased production at the vicinity of lung microvasculature; (2) a possible trapping in cell aggregates, as reported in coronary diseases (48); or (3) an eventual lung sequestration. Although the study does not provide data regarding an eventual direct effect of procoagulant microparticles on pulmonary vascular remodeling, the possible sequestration of microparticles supported by endothelium damage and inflammation is in accordance with an eventual thrombus formation and the altered pulmonary vasomotion observed in PAH.

The clinical data in the present study strengthen the hypothesis of noxious pathways involving procoagulant microparticles in the pathogenesis of severe PAH: the gradient of procoagulant microparticles is linked to mPAP, and circulating amounts of microparticles bearing active TF are inversely correlated to functional status (6MWD).

Regardless of their procoagulant properties, it is tempting to speculate that microparticles circulating in the pulmonary vascular bed could also contribute to lung injury through multiple and intricate pathways, including impaired perfusion, vascular remodeling, inflammatory response, and leukocyte recruitment. A variety of modulators of cell–cell cross-talk are borne by microparticles and could favor leukocyte infiltration and angiogenesis (49, 50). Furthermore, platelet-derived microparticles could promote the proliferation of smooth muscle cells and target vascular narrowing (51). Microparticles could also alter vasomotion through the following: (1) the delivery of thromboxane A2, a potent regulator of the vascular tone (52); (2) the inhibition of endothelial nitric oxide synthase expression (53); (3) the induction of oxidative stress (54); (4) the reduction of nitric oxide bioavailability (55).

Study Limitations
The control subset refers to patients with atypical chest pain and normal cardiovascular background and should be considered as a reference for comparison purposes. No comparison can be done between microparticle measurements obtained by capture onto annexin-V and antibodies because the affinity for both ligands is not similar. Annexin-V is a highly specific probe for PhtdSer with a disassociation constant of 10–14 mol/L, whereas the best antibodies allow interactions with a disassociation constant of about 10–9 mol/L. This is why studies on microparticle phenotypes should always compare clinical subsets to determine the relevance of the observed microparticle level variations.

No significant elevation in endothelium-derived microparticles expressing CD31, E-selectin, or fractalkine could be evidenced. This observation may indicate detection limits in the measurement of these endothelial markers in PAH, which depend on the following: (1) the level of endothelial markers borne by microparticles, (2) the antibody affinity, (3) the availability of the antigen (steric hindrance or limited proteolysis leading to an unrecognized epitope).

In conclusion, endothelial damage, neurohormonal activation, and procoagulant microparticles are related to the severity of PAH. Higher levels of microparticles bearing the active form of TF were measured in patients with PAH with lesser functional capacity, suggesting a possible prothrombotic role. The specific elevation in circulating amounts of procoagulant endothelium-derived microparticles expressing endoglin, a transmembrane receptor involved in endothelial cell proliferation, and the observation of a microparticle gradient across the precapillary lung circulation point at the endothelium as a key actor in the pulmonary microvasculature. Whether procoagulant microparticles play a major role in thrombus formation in PAH and subsequently contribute to the progression of the disease remains to be established.


   Acknowledgments
 
The authors thank Janet M. Thompson for careful reading of the manuscript.


   FOOTNOTES
 
Supported in part by a fellowship from the Comité National des Maladies Respiratoires, and institutional grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Université Louis Pasteur Strasbourg, and the Université Paris-Sud 11. O.M. was supported by a grant from the Fédération Française de Cardiologie.

* These authors contributed equally to this article and share first authorship. Back

** These authors are joint last authors. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200706-840OC on November 15, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 7, 2007; accepted in final form November 14, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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