Cleaved secretory leucocyte protease inhibitor as a biomarker of chymase activity in allergic airway disease.

BACKGROUND: Secretory leucocyte protease inhibitor (SLPI), which is present in many physiological fluids including saliva, sputum and nasal discharge, is the most effective inhibitor of chymase. Previously, we demonstrated that chymase is able to cleave SLPI and that the cleaved portion, cSLPI, is a biomarker of chymase activity. OBJECTIVE: We investigated the potential of cSLPI as a biomarker of chymase activity in subjects with allergic rhinitis (AR) and asthmatic airway disease. METHODS: Baseline sputum samples were collected from atopic asthmatics and healthy controls (HC). Nasal lavages (NAL) were performed in subjects with AR both at baseline and following a nasal challenge with allergen or placebo. Levels of cSLPI and chymase were determined by Western analysis, and tryptase and alpha-2 macroglobulin were measured by immunoassay. RESULTS: As compared with HC, asthmatics showed a significant increase in baseline cSLPI/total SLPI ratios and an increase in chymase levels. There was a high correlation of cSLPI/SLPI ratios to chymase levels in normal individuals and untreated asthmatics. In the NAL of patients with AR, as compared with placebo, allergen challenge increased inflammatory biomarkers, including cSLPI/SLPI ratios, chymase levels, tryptase levels and alpha2-macroglobulin levels. Correlations were observed between cSLPI/SLPI ratios and chymase levels and cSLPI/SLPI ratios and alpha2-macroglobulin levels; no correlation was seen between cSLPI/SLPI ratios and tryptase levels. CONCLUSION: Our data indicate that cSLPI reflects chymase activity in AR and asthma. Hence, cSLPI may serve as a biomarker for disease activity and for monitoring the efficacy of novel anti-inflammatory treatments in chymase-mediated diseases.

The future of anti-EGFR therapy.

We report here on the state of our knowledge of the target--namely, the epidermal growth factor (EGF) and its receptor--and the challenges related to the methods of determination of the epidermal growth factor receptor (EGFR) and associated molecular pathways. A critical review of the anti-EGFR therapeutic strategies is also outlined. The chimeric anti- EGFR monoclonal antibody cetuximab has been approved for EGFR-expressing colorectal tumors in patients who progress after irinotecan-based chemotherapy in combination with irinotecan and in squamous cell head and neck carcinomas for patients with locally advanced disease in combination with radiation therapy or after failure of platinum-based chemotherapy in recurrent or metastatic disease (FDA). Cetuximab has the potential to provide an improvement of clinical outcome also in other indications and tumor types, particularly when used as first-line therapy combined with standard chemotherapy for metastatic disease or in the adjuvant setting. Possible strategies to improve the effectiveness of anti-EGFR agents are suggested and include (i) the use of predictive tools capable of making a more rational selection of patients; (ii) the development of standardized predictive biomarkers as surrogates for early monitoring of drug efficacy; and (iii) adequate study design, statistical analysis and proper end points of efficacy to be applied in future prospective trials.

Clinical usefulness of bisphosphonates in oncology: treatment of bone metastases, antitumoral activity and effect on bone resorption markers.

The present article overviews the role of bisphosphonates for the treatment and prevention of bone metastases and their antiangiogenic effects and antitumoral activity. The skeleton is a frequent and clinically relevant site of metastasis in cancer patients. The major events related to bone metastases include bone pain, bone loss, hypercalcemia, spinal cord compression, and fractures. On the basis of their radiographic features, bone metastases are classified as osteoblastic, osteoclastic, or mixed. The primary goals of treatment of bone metastases are reduction of the risk of pathological fractures and other skeletal-related events, and pain control. Bisphosphonates are used to prevent pathological fractures by inhibition of osteoclasts. Recent studies suggest that bisphosphonates have some direct antitumoral activity, mainly mediated through the blockade of angiogenic pathways. Further clinical studies are needed to determine the optimal treatment duration, timing and schedule of bisphosphonates, assess their role as adjuvant therapy for the prevention of bone metastases, and establish their antiangiogenic activity in association with standard cytotoxic and hormonal drugs for treatment of patients with advanced disease.

MAP2 IHC detection: a marker of antigenicity in CNS tissues.

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer's disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.

Sequential adjuvant hormone therapy in postmenopausal breast cancer: rationale and clinical results.

For the past 15 years tamoxifen has been the standard adjuvant hormone therapy for women with early-stage breast cancer and estrogen receptor (ER)-positive tumors, irrespective of nodal status and other clinicopathological parameters. Recent studies provided evidence that the optimal duration of tamoxifen treatment is 5 years. Based on the positive clinical results obtained with the administration of aromatase inhibitors (AIs) in the metastatic setting, several controlled clinical trials have evaluated the efficacy and side effects of AIs versus standard tamoxifen also as adjuvant therapy in postmenopausal breast cancer patients. The results of the above studies, suggest a therapeutic advantage of AIs over tamoxifen with regard to relapse-free survival and the risk of metachronous contralateral breast cancer. We review the rationale and the available clinical data on initial or sequential hormone treatment with AIs and we propose a novel scenario for possible therapeutic strategies based on the clinicopathological characteristics of the patients and on the biology of each single tumor.

Aging and detoxifying enzymes responses to hypoxic or hyperoxic treatment.

We studied the levels of antioxidant and detoxifying enzymes in the livers and lungs of young and old rats kept under hypoxic or hyperoxic conditions as models of oxidative stress. In particular, we investigated the levels of enzymes directly involved in active oxygen species scavenging (superoxide dismutase, catalase and glutathione peroxidase-selenium dependent) and enzymes challenged with detoxification processes (glutathione transferase, glyoxalase I and glutathione reductase) in order to obtain a wide comparative view of the defence strategies used with respect to the age of the animals. The results show that the responses of some protective enzymes in young rats are opposite to those of old ones. Some of the changes found appear mainly due to age, while others appear to be due only to the oxygen tensions and are independent of the aging process. The glutathione contents of the liver and lung from young and old rats under hypoxic and hyperoxic conditions were measured.

Intracellular accumulation of beta-amyloid(1-42) in neurons is facilitated by the alpha 7 nicotinic acetylcholine receptor in Alzheimer's disease.

Amyloid beta(1-42), a major component of amyloid plaques, binds with exceptionally high affinity to the alpha 7 nicotinic acetylcholine receptor and accumulates intracellularly in neurons of Alzheimer's disease brains. In this study, we investigated the possibility that this binding plays a key role in facilitating intraneuronal accumulation of amyloid beta(1-42). Consecutive section immunohistochemistry and digital imaging were used to reveal the spatial relationship between amyloid beta(1-42) and the alpha 7 receptor in affected neurons of Alzheimer's disease brains. Results showed that neurons containing substantial intracellular accumulations of amyloid beta(1-42) invariably express relatively high levels of the alpha 7 receptor. Furthermore, this receptor is highly co-localized with amyloid beta(1-42) within neurons of Alzheimer's disease brains. To experimentally test the possibility that the binding interaction between exogenous amyloid beta(1-42) and the alpha 7 receptor facilitates internalization and intracellular accumulation of amyloid beta(1-42) in Alzheimer's disease brains, we studied the fate of exogenous amyloid beta(1-42) and its interaction with the alpha 7 receptor in vitro using cultured, transfected neuroblastoma cells that express elevated levels of this receptor. Transfected cells exhibited rapid binding, internalization and accumulation of exogenous amyloid beta(1-42), but not amyloid beta(1-40). Furthermore, the rate and extent of amyloid beta(1-42) internalization was related directly to the alpha 7 receptor protein level, since (1) the rate of amyloid beta(1-42) accumulation was much lower in untransfected cells that express much lower levels of this receptor and (2) internalization was effectively blocked by alpha-bungarotoxin, an alpha 7 receptor antagonist. As in neurons of Alzheimer's disease brains, the alpha 7 receptor in transfected cells was precisely co-localized with amyloid beta(1-42) in prominent intracellular aggregates. Internalization of amyloid beta(1-42) in transfected cells was blocked by phenylarsine oxide, an inhibitor of endocytosis. We suggest that the intraneuronal accumulation of amyloid beta(1-42) in Alzheimer's disease brains occurs predominantly in neurons that express the alpha 7 receptor. In addition, internalization of amyloid beta(1-42) may be facilitated by the high-affinity binding of amyloid beta(1-42) to the alpha 7 receptor on neuronal cell surfaces, followed by endocytosis of the resulting complex. This provides a plausible explanation for the selective vulnerability of neurons expressing the alpha 7 receptor in Alzheimer's disease brains and for the fact that amyloid beta(1-42) is the dominant amyloid beta peptide species in intracellular accumulations and amyloid plaques.

Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.

Increased expression of protease activated receptor-2 (PAR-2) in balloon-injured rat carotid artery.

Protease-induced cell signaling is mediated by specific receptors such as the emerging family of protease activated receptors (PARs). Since proteases are involved in various aspects of vascular injury, we assessed expression of PAR-2, a protease-activated receptor closely related to the thrombin receptor (PAR-1) but activated by an unknown protease, in vascular injury. Rat carotids were subjected to balloon-catheter injury and perfusion fixed at 1, 3, 7 or 14 days after injury. Sections of injured and normal carotid arteries were immunohistochemically labeled with a polyclonal antibody raised against the N-terminal residues 37-53 of human PAR-2. Sections were also labeled with antibodies to factor VIII-related antigen, smooth muscle actin and a proliferating cell nuclear antigen (PCNA). In normal vessels, PAR-2 labeling was diffuse and patchy in medial smooth muscle and endothelium. At one and three days after injury, before appearance of neointima, PAR-2 labeling increased in cells adjacent to damaged or necrotic smooth muscle cells. In addition, proliferating adventitial myofibroblasts labeled strongly for PAR-2. At 7 and 14 days after injury, the media and neointima of injured vessels had increased PAR-2 labeling which was most intense at the luminal edge of the neointima. Double immunohistochemical labeling confirmed the greatest expression of PAR-2 in areas with the greatest density of PCNA-positive cells. In addition, PAR-2 mRNA localization using in situ hybridization paralleled PAR-2 expression. The data suggest an upregulation of PAR-2 in response to vascular injury which is associated with medial smooth muscle damage, proliferating adventitial myofibroblasts and smooth muscle cells of the neointima, particularly those at the proliferating luminal edge of the neointima. Possible functional consequences of this receptor upregulation and its role in the response to vascular injury remain to be determined.

Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism.

Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.

Vanilloid receptor 1 antagonists attenuate disease severity in dextran sulphate sodium-induced colitis in mice.

Neurogenic mechanisms have been implicated in the induction of inflammatory bowel disease (IBD). Vanilloid receptor type 1 (TRPV1) has been visualized on nerve terminals of intrinsic and extrinsic afferent neurones innervating the gastrointestinal tract and local administration of a TRPV1 antagonist, capsazepine, reduces the severity of dextran sulphate sodium (DSS)-induced colitis in rats (Gut 2003; 52: 713-9(1)). Our aim was to test whether systemically or orally administered TRPV1 antagonists attenuate experimental colitis induced by 5% DSS in Balb/c mice. Intraperitoneal capsazepine (2.5 mg kg(-1), bid), significantly reduced the overall macroscopic damage severity compared with vehicle-treated animals (80% inhibition, P < 0.05); however, there was no effect on myeloperoxidase (MPO) levels. An experimental TRPV1 antagonist given orally was tested against DSS-induced colitis, and shown to reverse the macroscopic damage score at doses of 0.5 and 5.0 mg kg(-1). Epithelial damage assessed microscopically was significantly reduced. MPO levels were attenuated by approximately 50%, and diarrhoea scores were reduced by as much as 70%. These results suggest that pharmacological modulation of TRPV1 attenuates indices of experimental colitis in mice, and that development of orally active TRPV1 antagonists might have therapeutic potential for the treatment of IBD.

Abnormal patterns of microtubule-associated protein-2 (MAP-2) immunolabeling in neuronal nuclei and Lewy bodies in Parkinson's disease substantia nigra brain tissues.

Parkinson's disease (PD) is a neurodegenerative disorder associated with the appearance of cytoplasmic Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra and the progressive loss of these neurons. Cytoskeleton alterations and associated impairments of neuronal transport may contribute to neuronal death. Microtubule-associated protein-2 (MAP-2), a cytoskeleton protein is localized primarily in neuronal dendrites and is known to stabilize microtubule assembly and mediate their interactions with other neuronal cell components. To determine if alterations in MAP-2 morphology are present in PD neurons, we used single and double immunohistochemical and immunofluorescent techniques to characterize MAP-2 in PD neuronal tissues. We report abnormal MAP-2 immunolabeling in some neurons of the substantia nigra of PD brain tissues, which were not observed in the normal, age-matched, control brain tissues. Furthermore, MAP-2 was co-localized with alpha-synuclein and ubiquitin in cytoplasmic LBs of neurons. Surprisingly, MAP-2 was also found to form fibrous aggregates and crystal-like structures within neuronal nuclei. These PD-associated alterations in MAP-2 morphology and distribution suggest that impaired neuronal transport may contribute to the progression of neuronal loss in the brains of PD patients.

Male breast carcinoma in situ. Report of a case diagnosed by nipple discharge cytology alone.

A case of male breast carcinoma is reported, whose only clinical sign was a serous nipple discharge. The presence in the cytologic smears of atypical epithelial cells organized in a papillary structure suggested a papillary neoplasia, which at the histologic examination was found to be a ductal carcinoma in situ.

Nephrotic syndrome and adjuvant treatment with tamoxifen for early breast cancer. Case report and review of the literature.

A 56 year old postmenopausal patient underwent a right radical mastectomy for a stage I breast cancer. An adjuvant treatment with tamoxifen was planned, but, because of patient's choice, a regular administration of the endocrine treatment started only one year after the operation. After a very short period of drug administration, she developed a steroid responsive nephrotic syndrome. We have reviewed the international literature concerning drug-induced renal disease; this is the first case of a tamoxifen-induced nephrotic syndrome.

Primary lymphoma of the breast: a case report.

The clinical features of a patient with a primary lymphoma of the breast are herein reported. The diagnosis was reached by histological examination after outpatient surgery. Surgical resection was followed by cytostatic treatment and locoregional radiotherapy. This case report is an example where the integration of physical and mammographic examination together with ultrasonography (and cytology), in the context of diagnostic procedures, induced us to perform a surgical excision on an outpatient basis. This line of action allowed us, in one step, to arrive at both the definitive diagnosis and the appropriate choice of therapy. Thus we believe that this diagnostic procedure should be carried out whenever a breast lesion, thought to be "probably benign", is found by physical or mammographic exam in a peri-postmenopausal woman.

Ultrasound in the detection of breast cancer associated with isolated clustered microcalcifications, mammographically identified.

The purpose of this work was to evaluate the effectiveness of US scanning in detecting breast cancer associated with isolated clustered microcalcifications (MC) mammographically identified. 52 isolated clusters of MC mammographically detected underwent histological examination. In all cases, ultrasonographic of (US) and cytologic examinations were performed for the improved definition of the nature of the MC. Fine needle aspiration was performed under radiographical (Rx)-guidance, only in cases of US-negativity. Ultrasound detected a breast lesion in 31 cases, out of which 24 (77.4%) were found to be carcinomas (invasive in 87.5% of the cases). Out of the remaining 21 US-negative cases, 7 (33.3%) were malignant lesions (15% invasive). Statistical analysis demonstrated that US-positivity significantly correlated with the presence of malignant lesions (p < 0.001). US-examination should also be performed in cases of isolated clustered microcalcifications for a more accurate selection of cases requiring histological verification.

The role of cytology in differentiation of breast lesions.

The introduction of cytologic examination in to the diagnostic procedure has made it possible to define breast lesions better as early as the preoperative stage. However, there are interpretative problems depending on the nature of the lesions that make a histologic examination necessary. The cytology of nipple discharge or FNA of breast lesions would permit the best possible selection of the cases, indicating not only whether surgery is necessary, but also the basis on which it should be performed (outpatient under local anesthesia or inpatient under general anesthesia). The definitive histologic diagnosis of 447 cases submitted to surgery under general anesthesia and of 379 who were operated under local anesthesia has been correlated with the relative cytologic examination. The sensitivity and specificity of the cytologic examination are respectively 97.7% and 98.8%.

Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology.

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.

MAP-2 immunolabeling can distinguish diffuse from dense-core amyloid plaques in brains with Alzheimer's disease.

Alzheimer's disease (AD) neuropathology is characterized by the presence of diffuse and dense-core (neuritic) amyloid plaques in specific areas of the brain. The origin of these plaques and the relationship between them is poorly understood. Current methods to identify clearly these types of plaques in the AD brains are largely dependent upon morphological characteristics. Dense-core amyloid plaques in the entorhinal cortex and hippocampus of AD brains might arise from the lysis of neurons overburdened by excessive intracellular deposition of amyloid beta1-42 (Abeta42) peptide. The local release of active lysosomal enzymes, which persist within these plaques, might degrade most of the released intracellular proteins, leaving behind only those that are resistant to proteolytic digestion, such as ubiquitin, tau, neurofilament proteins and amyloid. To test the possibility that proteins that are sensitive to proteolysis may be degraded selectively in plaques, we used immunohistochemistry to examine the distribution of microtubule-associated protein-2 (MAP-2), a protein localized primarily in neuronal dendrites and known to be sensitive to proteolysis. Uniform MAP-2 immunolabeling was detected throughout the somatodendritic compartment of neurons in age-matched control cortical brain tissues as well as throughout areas of Abeta42-positive diffuse plaques in AD brains. In contrast, analysis of serial sections revealed that MAP-2 was absent from Abeta42-positive dense-core plaques in AD brains. Our results indicate that this differential MAP-2 immunolabeling pattern among plaques may be employed as a reliable and sensitive method to distinguish dense-core plaques from diffuse plaques within AD brain tissue. Furthermore, this biochemical distinction indicates that dense-core and diffuse plaques are formed by different mechanisms.

Localization of protease-activated receptors-1 and -2 in human mast cells: indications for an amplified mast cell degranulation cascade.

Protease-activated receptors (PARs) belong to a family of G-coupled seven transmembrane receptors that are activated by a proteolytic cleavage of their N-termini. Recent studies suggest the involvement of protease-activated receptors-1 and -2 (PAR-1, PAR-2) activators in mast cell degranulation in various physiological and pathophysiological processes in inflammatory responses. Although PAR-1 and PAR-2 activating proteases, thrombin and tryptase, have been associated with mast cell activation, PAR-1 and PAR-2 have not been localized within these cells. We describe here the localization of PAR-1 and PAR-2 in mast cells from various normal human tissues using immunohistochemical and double immunofluorescence techniques. The presence of these receptors on the membrane may explain the actions of accessible extracellular thrombin and tryptase for mast cell activation. In addition to the membrane labeling, these receptors are also localized on the membrane of the intracellular tryptase-positive granules, which may function to sustain further mast cell degranulation upon exocytosis. The localization of these two receptors in mast cells suggests a novel mechanism for controlling mast cell activation through regulation of PAR-1 and PAR-2.