This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Lucas, M. J. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Lucas, M. J.

Expression of Thrombin Receptors in Endothelial Cells and Neutrophils from Normal and Preeclamptic Pregnancies

Departments of Obstetrics and Gynecology (Y.W., Y.G., M.J.L.) and Cellular and Molecular Physiology (Y.W.), Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130

Address all correspondence and requests for reprints to: Yuping Wang, M.D., Ph.D., Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center, P.O. Box 33932, Shreveport, Louisiana 71130. E-mail: . ywang1{at}lsuhsc.edu

Abstract

Thrombin receptors, i.e. proteinase-activated receptors (PARs), are expressed in endothelial cells (ECs) and neutrophils and directly affect platelet function and thrombosis. Although endothelial dysfunction and neutrophil activation have been demonstrated in women with preeclampsia (PE), the expression and regulation of PARs have not been defined in PE. In this study, we measured the expression of PARs in ECs and in neutrophils derived from normal and preeclamptic pregnancies. We also examined the effects of placental factors on PAR expression in these cells in vitro. ECs were isolated from umbilical cords (human umbilical vein ECs) from normal and preeclamptic pregnancies. Neutrophils were isolated from blood obtained from nonpregnant, uncomplicated pregnant, and preeclamptic women. Total RNA was extracted from the first-passage (P1) ECs (normal and PE) and from normal P1 ECs incubated with conditioned media derived from normal and preeclamptic placental cultures. The mRNA expression of thrombin receptor (PAR1), PAR2, and PAR3 was measured by ribonuclease protective assay. The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for each sample. We found that: 1) PAR1 expression was enhanced in ECs from PE, compared with ECs from normal pregnancies; 2) PAR2 expression was expressed in PE ECs but not in normal ECs; 3) neutrophils from nonpregnant women, normal, and preeclamptic pregnancies expressed PAR2, whereas only neutrophils from normal and preeclamptic pregnancies expressed PAR1; and 4) factors released from preeclamptic placenta up-regulated PAR1 and PAR2 expression in ECs but not in neutrophils. We conclude that mRNA expression of PAR1 and PAR2 is increased in ECs derived from preeclamptic pregnancies. Up-regulation of thrombin receptor expression in neutrophils may be a unique phenomenon during pregnancy but not apparently unique to PE. Factors released from the placenta are likely candidates in regulating PAR expression in ECs and may contribute to the platelet activation and vascular endothelial dysfunction in PE.

THROMBIN RECEPTORS, i.e. proteinase-activated receptors (PARs), are G protein-coupled membrane receptors, and they are expressed on a variety of cell types, such as endothelial cells (ECs), platelets, fibroblasts, monocytes, and T cells. There are three known members of this receptor family: thrombin receptor (PAR-1), PAR-2, and PAR-3. PAR-1 and PAR-3 are cleaved and activated by thrombin, and PAR-2 is cleaved and activated by trypsin-like protease. PARs have diverse effects on ECs and platelets. PARs expressed on endothelium interact dynamically with circulating components to maintain a delicate balance between anticoagulant and procoagulant activities. An intact endothelial layer normally inhibits clot formation by constitutively expressing the antithrombolic glycoprotein thrombomodulin on the cell surface. In addition to the critical role of PARs and thrombin in platelet aggregation, many biological actions of PARs are also related to inflammation. PARs mediate vasodilation and vasoconstriction, vascular permeability, and adhesion and infiltration by chemotactic cells (1).

Endothelial dysfunction and platelet activation has been demonstrated in women with preeclampsia (PE), a multisystem disorder unique to human pregnancy. Pathophysiologically, PE is characterized by increased vasoconstriction, increased vascular permeability and increased platelet aggregation leading to maternal hypertension, proteinuria and thrombocytopenia, respectively. Maternal vascular system also experience inflammatory process as evidenced by increased neutrophil and complement activation in this pregnancy disorder. Thrombin levels are significantly increased in maternal circulation during PE. Thrombin alters EC interactions and increases endothelial monolayer permeability in vitro (2). In contrast, antithrombin III, a native thrombin inhibitor, as well as thrombomodulin are depleted in PE (3). The protease thrombin activates platelets, leukocytes and ECs and is formed at sites of coagulation and inflammation. These important proinflammatory cellular events are relevant to the pathophysiology of PE.

The role of thrombin and its receptor family in the physiologic and pathophysiologic processes lead us to study PARs expression in ECs and neutrophils derived from normal and preeclamptic pregnancies. We also tested the effects of placental factor(s) on PAR expression. In this study, we examined: 1) the expression of PARs in ECs isolated from normal and preeclamptic pregnancies; 2) expression of PARs in neutrophils isolated from nonpregnant females, women from normal pregnancy and complicated with PE; and 3) regulatory effects of placental factor(s) on PARs expression in ECs and neutrophils.

Materials and Methods

Placentas and umbilical cords were obtained immediately after delivery from normal and preeclamptic pregnancies (as defined by American College of Obstetrics and Gynecology). Normal pregnancy is defined as pregnancy in a woman with normal blood pressure of less than 140/90 mm Hg, no proteinuria, and no medical or obstetrical complications. Mild PE is defined as a maternal blood pressure of 140/90 mm Hg or higher, with the presence of proteinuria (300 mg/24 h), after 20 wk of gestation. Severe PE is defined as a maternal blood pressure of 160/110 mm Hg or higher on two separate readings, at least 6 h apart, with proteinuria. Human neutrophils were isolated from freshly drawn venous blood from nonpregnant healthy female volunteers who were free of any chronic medical illnesses and from women with normal and preeclamptic pregnancies. Table 1 and Table 2 show the demographics for the women from whom the samples were obtained. We did not include blood pressure and proteinuria in the tables because preeclamptic patients in this study were all clinically diagnosed as severe PE. This study was approved by the Institutional Review Board for Human Research at Louisiana State University Health Sciences Center in Shreveport.

HUVECs were isolated from human umbilical cords by collagenase treatment as previously described (4, 5). The cells were placed on a fibronectin (25 µg/ml)-coated 25-cm² culture flask and incubated with EC growth medium (BioWhittaker, Inc., Walkersville, MD). Media were changed within 24 h of isolation and then every 3 d. When cells were confluent, they were harvested with 0.01% trypsin/EDTA (Sigma; St. Louis, MO) and placed into a 75-cm² cell culture flask. All cell culture surfaces were coated with fibronectin (Biomedical Technologies, Stoughton, MA). When the monolayer cells reached confluence, total RNA was isolated and used for determination of mRNA expression. Only the primary passage (P1) of ECs was used in experiments.

Neutrophil isolation

Human neutrophils were isolated by using the method previously described (5, 6, 7): dextran sedimentation and Histopaque density gradient centrifugation, followed by lysis of contaminating red blood cells. In general, this procedure yielded approximately 1–2 x 10⁷ neutrophils from 10 ml whole blood, with 99% viable by trypan blue exclusion and 98% pure by acetic acid-crystal violet staining. Freshly isolated neutrophils were immediately used for the total RNA extraction. RNA samples were stored at -70 C until assay.

Preparation of placental-conditioned medium

Placental-conditioned media were prepared by culturing placental villous tissue for 48 h (8, 9). Briefly, fresh placentas from normal and preeclamptic pregnancies were processed immediately after delivery. Placental tissue was gently separated by sterile dissection from different cotyledons, excluding chorionic and basal plates, minced with scalpel blades, and washed repeatedly with PBS to remove blood from the intervillous space. Whole villous tissue was incubated with serum free DMEM (Sigma). The incubation was carried out for 48 h at 37 C in an incubator gassed with 95% air-5% CO₂ (Forma Scientific, Inc., Marietta, OH). Medium samples were collected at the end of incubation as conditioned medium and stored at -80 C until assay.

Total RNA isolation and mRNA expression by ribonuclease (RNase) protection assay (RPA)

Total RNA was isolated from ECs and neutrophils by acid guanidine thiocyanate phenol chloroform extraction (Tri Reagent; Molecular Research Center, Inc., Cincinnati, OH). The mRNA expression of thrombin receptor (PAR1), proteinase-activated factor-2 (PAR2), and 3 (PAR3) was examined by RPA. RiboQuant multiprobe RPA system was purchased from PharMingen (San Diego, CA). The RPA system includes multiprobe template sets, an in vitro transcription kit, and an RPA kit. We choose the template of human Angio-2 from PharMingen. Besides PAR1, PAR2, and PAR3, this template also includes endothelin receptor-, endothelin receptor-ß, endothelin receptor-ß-like, platelet-activating factor receptor (PAFR), H963PAF receptor, PAFR-like, L32, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). For RPA, the probes were labeled with [-³²P]uridine 5'-triphosphate (3000 Ci/mmol, 10 mCi/ml), and total RNA of 5 µg from each sample was used in each reaction. The labeled probes and sample RNA were incubated under conditions that favor hybridization of complementary sequences. After hybridization, the mixture was treated with RNase to degrade unhybridized probes. The labeled probes that were hybridized to complementary RNA from the sample were then protected from RNase digestion and were separated on a polyacrylamide gel for autoradiography. Because each unprotected probe includes flanking sequences derived from the multiple cloning site of the plasmid, the length of protected probe or sample is 29 nucleotides shorter than the length of unprotected probe. Therefore, protected probe (sample) migrates further on a polyacrylamide gel, as observed on autoradiography. The nucleotide length for unprotected and protected probes is listed in Table 3. The template contains two housekeeping genes, L32 and GAPDH. The assay procedure followed the manufacturer’s instructions (PharMingen).

Patient clinical characteristics for endothelial samples are shown in Table 1, and those for neutrophil samples are shown in Table 2. Tables 1 and 2 include racial status, parity, gestational age at delivery, mode of delivery, infection, birth weight, and birth weight percentiles. We have not seen any pattern of relationships of these variables and our results.

Endothelial cells

Figure 1 shows the mRNA expression of PARs in ECs isolated from five normal and four preeclamptic pregnancies. ECs from PE expressed more PAR-1 than ECs from normal pregnancies. PAR-2 expression was not detected in normal ECs but was expressed in ECs from PE. Neither normal ECs nor PE ECs expressed PAR-3. In addition, mRNA expression of endothelin receptor group (endothelin receptor-, endothelin receptor-ß, and ß-like) and PAFR group (PAFR, H963PAF receptor, and PAFR-like) was not detectable in these EC samples from normal and preeclamptic pregnancies.

View larger version (74K): [in this window] [in a new window]   Figure 1. The mRNA expression of PARs in ECs isolated from normal pregnancy and from PE determined by RPA. Total RNA of 5 µg per sample was used in each reaction. Lanes 1–5, Normal ECs; lanes 6–9, PE ECs. Endothelial cells from PE express more thrombin receptor (PAR-1) and PAR-2 than normal ECs. PAR-3 expression is not detectable in ECs from normal pregnancies and from women with PE.

The mRNA expression of PARs in neutrophils from five nonpregnant healthy females, six from women with normal pregnancies, and six from women with PE was examined. Representative results are shown in Fig. 2. PAR1 was not expressed in neutrophils from nonpregnant females but was expressed in neutrophils from normal pregnancy and from PE. PAR-2 was expressed in neutrophils from all three groups, whereas none of the neutrophil samples expressed PAR-3. The mRNA expression for endothelin receptors was not detectable in neutrophils from all three groups; whereas mRNA expression for PAFR and H963PAF receptor was strongly expressed in all neutrophils.

View larger version (104K): [in this window] [in a new window]   Figure 2. A representative mRNA expression of PARs in neutrophils isolated from nonpregnant females, women with normal pregnancy, and women with PE. Total RNA of 5 µg per sample was used in each reaction. Lanes 1–3, Neutrophils from nonpregnant females; lanes 4–6, neutrophils from normal pregnancies; lanes 7–9, neutrophils from PE. In addition to PARs, PAFR and H963PAFR are also expressed in neutrophils in all three groups.

To test whether factor(s) released by placentas could regulate PAR expression in ECs, P1 ECs were exposed to conditioned medium derived from placental villous tissue culture. In this experiment, ECs isolated from normal pregnancies were used, and isolated normal ECs (usually from four to five individuals) were pooled together in culture for each assay. When these combined P1 normal ECs reached confluence, they were treated with placental-conditioned media for 15 min, 30 min, 1 h, 2 h, and 4 h. The placental-conditioned media were also pooled either from three to five normal or from three to five preeclamptic placental cultures for each experiment. Figure 3 shows a representative mRNA expression in normal ECs treated with normal or preeclamptic placental-conditioned media. We found no change in PAR1 expression in ECs after exposure to normal placental-conditioned media. PAR2 expression was up-regulated only with longer time exposure (4 h) to normal placental-conditioned media (Fig. 3). Compared with ECs exposed to normal placental-conditioned media, ECs exposed to preeclamptic placental-conditioned media showed dramatic up-regulation of both PAR1 and PAR2, and the up-regulation was time-dependent (Fig. 3). These results demonstrated a differential response of ECs to factors derived from normal and preeclamptic placentas.

View larger version (78K): [in this window] [in a new window]   Figure 3. The mRNA expression of PARs in ECs exposed to conditioned media derived from normal placental tissue cultures and from preeclamptic placental tissue cultures for 15 min, 30 min, 1 h, 2 h, and 4 h. Pooled normal ECs were used, as well as pooled normal conditioned media or pooled preeclamptic conditioned media, as stated in Results. Total RNA of 5 µg per sample was used in each reaction. The mRNA expression for PAR-1 and PAR-2 is dramatically up-regulated in ECs exposed to preeclamptic conditioned media (CM), compared with ECs exposed to normal placental CM. The results are representative of three independent experiments. C, Control ECs.

View larger version (63K): [in this window] [in a new window]   Figure 4. The mRNA expression of PARs in neutrophils exposed to conditioned media derived from preeclamptic placental tissue cultures (PECM) for 5 min and 15 min. Total RNA of 5 µg per sample was used in each reaction. PAR-2 expression and PAFR expression are up-regulated in neutrophils after the exposure. The result is representative of three independent experiments.

In this study, we found that mRNA expression of thrombin receptor and PAR2 is up-regulated in ECs isolated from preeclamptic pregnancies, compared with ECs isolated from normal pregnancies. PAR3 is not expressed in these EC samples. Our findings of up-regulation of expressions for thrombin receptor and PAR2 in ECs from PE are consistent with observed increases in coagulation activity and platelet activation in women with PE. How thrombin receptor and PAR2 became up-regulated in ECs from PE is unknown. To test the hypothesis that soluble factors released from the placenta may be etiologic candidates for this event in PE, we exposed ECs to placental-conditioned media in our in vitro culture system. We found that placental factor(s) stimulates mRNA expression of thrombin receptor and PAR2 in ECs. This effect is dramatically increased in ECs exposed to conditioned media from preeclamptic placental cultures. These findings suggest that placenta from PE produces or releases more components that exert protease activities, than that of normal placenta does.

Increased thrombin receptor and PAR2 gene expression in ECs from PE and up-regulation of these gene expressions by factors released by placentas from PE indicate that thrombin receptor and PAR2 up-regulation may be an event of altered endothelial function in PE. It is known that altered endothelial function (such as increased vasoconstriction, increased endothelial permeability, and platelet aggregation) plays an important role in the pathophysiology of PE. A recent study showed that PAR2-activating peptides can cause vasoconstriction in rat vascular endothelium and in intact human umbilical vein rings (10). PAR2-activating peptides can also induce leukocyte rolling, adhesion, and extravasation (11). Further, activation of PAR2 induces rapid formation of tissue factor mRNA in ECs, and this effect is accompanied by an increased tissue factor activity at the EC surface and promotes blood coagulation in vitro (12). We previously reported that neutrophil-endothelial adhesion is increased in ECs derived from PE (5). We also found that factors produced by placentas from PE can induce neutrophil-endothelial adhesion in vitro and regulate neutrophil integrin CD62L and CD11b expression (8, 9). Therefore, increased thrombin receptor and PAR2 expression in ECs from preeclamptic pregnancies may be a part of the altered endothelial function that characterizes PE and contributes to the pathophysiology of this pregnancy disorder.

Compared with ECs, we found that neutrophils express PAR2 in all samples, whether isolated from nonpregnant females, normal pregnancies, or pregnant women with PE. However, we found thrombin receptor expressed only in neutrophils isolated from pregnant women but not from nonpregnant subjects. Expression of PAR2 in neutrophils has been demonstrated (13). The information about thrombin receptor (PAR1) expression in neutrophils has not been described. It is known that thrombin, acting thought PAR-1, affects a wide range of physiologic activities (14). Thrombin is required to initiate platelet aggregation. Thrombin has also been shown to alter EC interactions and increase endothelial monolayer permeability associated with changes in cell-cell junctional organization (2, 15). Thrombin levels are significantly increased in maternal circulation during PE. In contrast, antithrombin III, a native thrombin inhibitor, is depleted in PE (3). The finding that thrombin receptor expression is increased in ECs from preeclamptic pregnancies and that thrombin receptor is expressed in neutrophils during pregnancy suggests that thrombin receptor may play an important role in pro- and antiaggregation processes in cellular communications between ECs, platelets, and neutrophils during pregnancy.

One question that arises from our study is whether up-regulation of thrombin receptor expression in neutrophils is labor-induced, given that only neutrophils from pregnant women express thrombin receptor. In our study, neutrophil samples were obtained from both vaginal and cesarean deliveries, before and after onset of labor, in both normal and preeclamptic groups. Neutrophils obtained from normal pregnant women at 32–33 wk gestation also expressed thrombin receptor. Therefore, it is not likely that expression of thrombin receptor in neutrophils during pregnancy is labor-induced or is attributable to the difference in gestational age. Similarly, it is not likely that infection or inflammatory process is the cause, even though one patient in the preeclamptic group had chorioamnionitis. Our finding of expression of thrombin receptor in neutrophils from normal and from preeclamptic pregnancies suggests that up-regulation of thrombin receptor expression in neutrophils may be a unique phenomenon during pregnancy. The role of neutrophil up-regulation of thrombin receptor expression during pregnancy is not known. It is possible that neutrophils are a part of sequential mechanisms involved in promoting the coagulation process during pregnancy. Another possibility is that up-regulation of thrombin receptor expression in neutrophils is a part of compensatory mechanisms in the coagulation process to neutralize increased thrombin levels in the maternal circulation during pregnancy. To further investigate whether factors, released from the placenta, are responsible for the up-regulation of neutrophil thrombin receptor expression as placental factors to ECs, neutrophils were treated with placental-conditioned media. Unexpectedly, up-regulation of thrombin receptor expression was not observed. These results suggest that factors present in the maternal circulation other than placental sources may account for the up-regulation of neutrophil thrombin receptor during pregnancy. Whether or not hormonal changes during pregnancy influence thrombin receptor expression remains to be determined.

In this study, we found that PAR-3 expression was not detectable in either ECs or neutrophils. The physiological role of PAR-3 in human is not known (16). Although PAR-3 is considered a second thrombin receptor in humans (17), our results suggest that PAR-3 is not likely to be involved in the pathophysiology of PE.

PE is a clinically heterogeneous disorder. All subjects in the preeclamptic group had severe, preterm-onset disease, except one. Severe, preterm-onset PE may indeed be different from mild, term-onset disease. Nonetheless, the common elements related to endothelial dysfunction characterize all forms of PE. We do not think that our results reflect some confounding aspect of the clinical heterogeneity of PE. Endothelial dysfunction characterizes the entire spectrum of disease, and the placenta is the presumptive contributor of the dysfunction. The differential responses of the PAR receptors associated with PE also suggest a specific and valid relationship.

In summary, we found that expressions of thrombin receptor and PAR2 are increased in ECs from PE, compared with ECs from normal pregnancies. We also found that neutrophils express PAR2, but only neutrophils from pregnancies express thrombin receptor. Circulating placental factors contribute to the up-regulation of thrombin receptor and PAR2 expression in ECs from PE, whereas circulating factors other than placental origin may be responsible for the up-regulation of thrombin receptor in neutrophils during pregnancy. Because of the diverse biological effects of these PARs on the vascular system, further study of these PARs will likely help us to better understand the function of these molecules during pregnancy and their roles in PE.

Acknowledgments

Footnotes

This work was supported in part by Grants HD-36822 from The National Institute of Child Health and Human Development, HL-65997 from the National Heart, Lung, and Blood Institute, and LEQSF-RD-A13 from the Board of Regents Support Fund of Louisiana State. The results of this study were presented at the 48th Annual Meeting of The Society for Gynecologic Investigation, Toronto, Canada, March 14–17, 2001.

Abbreviations: EC, Endothelial cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HUVEC, human umbilical vein EC; PAFR, platelet-activating factor receptor; PAR, proteinase-activated receptor; PE, preeclampsia; RNase, ribonuclease; RPA, RNase protection assay.

Received December 19, 2001.

Accepted April 26, 2002.

References

1. Dery O, Corvera CU, Steinhoff M, Bunnett NW 1998 Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol 274:C1429–C1452
2. Rabiet MJ, Plantier JL, Rival Y, Genoux Y, Lampugnani MG, DeJana E 1996 Thrombin-induced increase in endothelial permeability is associated with changes in cell to cell junction organization. Arterioscler Thromb Vasc Biol 16:488–496[Abstract/Free Full Text]
3. Hsu CD, Johnson TRB, Hong SF, Chan D 1995 Altered circulating thrombomodulin levels and antithrombin-III activities as evidence for varied activation of the coagulation cascade in severely versus mildly preeclamptic pregnancies. J Matern Fetal Investig 5:140–143
4. Jaffe EA, Nachman RL, Becker CG, Minick CR 1973 Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756
5. Wang Y, Adair CD, Coe L, Weeks JW, Lewis DF, Alexander JS 1998 Activation of endothelial cells in preeclampsia: increased neutrophil-endothelial adhesion correlates with up-regulation of adhesion molecule P-selectin in human umbilical vein endothelial cells isolated from preeclampsia. J Soc Gynecol Invest 5:237–243[CrossRef][Medline]
6. Markert M, Andrews PC, Babior BM 1984 Measurement of O₂^- production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils. Methods Enzymol 105:358–365[Medline]
7. Ohno N, Ichikawa H, Coe L, Kvietys PR, Granger DN, Alexander JS 1997 Soluble selectins and ICAM-1 modulate neutrophil-endothelial adhesion and diapedesis in vitro. Inflammation 21:313–324[CrossRef][Medline]
8. Wang Y, Adair CD, Weeks JW, Lewis DF, Alexander JS 1999 Increased neutrophil-endothelial adhesion induced by placental factors is mediated by platelet-activating factor in preeclampsia. J Soc Gynecol Invest 6:136–141[CrossRef][Medline]
9. Wang Y, Gu Y, Philibert L, Lucas MJ 2001 Neutrophil activation induced by placental factors in normal and pre-eclamptic pregnancies in vitro. Placenta 22:560–565[CrossRef][Medline]
10. Saifeddine M, Roy SS, Al-Ani B, Triggle CR, Hollenberg MD 2000 Endothelium-dependent contractile actions of proteinase-activated receptor-2-activating peptides in human umbilical vein: release of a contracting factor via a novel receptor. Br J Pharmacol 125:1445–1454[CrossRef][Medline]
11. Vergnolle N 1999 Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo. J Immunol 163:5064–5069[Abstract/Free Full Text]
12. Alm A-K, Norstrom E, Sundelin J, Nystedt S 1999 Stimulation of proteinase activated receptor-2 causes endothelial cells to promote blood coagulation in vitro. Thromb Haemost 81:984–988[Medline]
13. Howells GL, Macey MG, Chinni C, Hou L, Fox MT, Harriott P, Stone SR 1997 Proteinase-activated receptor-2: expression by human neutrophils. J Cell Sci 110:881–887[Abstract]
14. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R 2001 Proteinase-activated receptors. Pharmacol Rev 53:245–282[Abstract/Free Full Text]
15. Garcia JGN, Verin AD, Schaphorst KL 1996 Regulation of thrombin-mediated endothelial cell contraction and permeability. Semin Thromb Hemost 22: 309–315
16. Vergnolle N, Wallace JL, Bunnett NW, Hollenberg MD 2001 Protease-activated receptors in inflammation, neuronal signaling and pain. Trends Pharmacol Sci 22:146–152[CrossRef][Medline]
17. Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, Coughlin SR 1997 Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386:502–506[CrossRef][Medline]

This article has been cited by other articles:

				V. Shpacovitch, M. Feld, M. D. Hollenberg, T. A. Luger, and M. Steinhoff Role of protease-activated receptors in inflammatory responses, innate and adaptive immunity J. Leukoc. Biol., June 1, 2008; 83(6): 1309 - 1322. [Abstract] [Full Text] [PDF]

				T. J. Moraes, R. Martin, J. D. Plumb, E. Vachon, C. M. Cameron, A. Danesh, D. J. Kelvin, W. Ruf, and G. P. Downey Role of PAR2 in murine pulmonary pseudomonal infection Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L368 - L377. [Abstract] [Full Text] [PDF]

				K. Hirano The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology Arterioscler. Thromb. Vasc. Biol., January 1, 2007; 27(1): 27 - 36. [Abstract] [Full Text] [PDF]

				V. M. Shpacovitch, G. Varga, A. Strey, M. Gunzer, F. Mooren, J. Buddenkotte, N. Vergnolle, C. P. Sommerhoff, S. Grabbe, V. Gerke, et al. Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices J. Leukoc. Biol., August 1, 2004; 76(2): 388 - 398. [Abstract] [Full Text] [PDF]

				Y. Wang, D. F. Lewis, Y. Gu, Y. Zhang, J. S. Alexander, and D. N. Granger Placental Trophoblast-Derived Factors Diminish Endothelial Barrier Function J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2421 - 2428. [Abstract] [Full Text] [PDF]

				T. Minami, A. Sugiyama, S.-Q. Wu, R. Abid, T. Kodama, and W. C. Aird Thrombin and Phenotypic Modulation of the Endothelium Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 41 - 53. [Abstract] [Full Text] [PDF]

				D. H. Sturn, N. C. Kaneider, C. Feistritzer, A. Djanani, K. Fukudome, and C. J. Wiedermann Expression and function of the endothelial protein C receptor in human neutrophils Blood, August 15, 2003; 102(4): 1499 - 1505. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Lucas, M. J. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Lucas, M. J.