Use of botulinum toxin type A in symptomatic accessory soleus muscle: first five cases.

Symptomatic accessory soleus muscle (ASM) can cause exercise-induced leg pain due to local nerve/vascular compression, muscle spasm, or local compartment syndrome. As intramuscular injections of botulinum toxin type A (BTX-A) can reduce muscle tone and mass, we investigated whether local BTX-A injections relieve the pain associated with symptomatic ASM. We describe five patients presenting peri/retromalleolar exertional pain and a contractile muscle mass in the painful region. Com-pression neuropathy was ruled out by electromyo-graphic analysis of the lower limb muscles. Doppler ultrasonography was normal, excluding a local vascular compression. ASM was confirmed by magnetic resonance imaging. After a treadmill stress test, abnormal intramuscular pressure values in the ASM, confirmed the diagnosis of compartment syndrome only in one patient. All five patients received BTX-A injections in two points of the ASM. The treatment efficacy was evaluated based on the disappearance of exercise-induced pain and the resumption of normal physical and sports activities. After BTX-A injection, exertional pain disappeared and all five patients resumed their normal level of physical and sports performances. Neither side effects nor motor deficits were observed. BTX-A is well tolerated in patients with ASM and could be used as a new conservative therapeutic strategy for the treatment of symptomatic ASM before surgery.

Total knee arthroplasty in severe haemophilic patients under continuous infusion of clotting factors.

PURPOSE: Haemophilic arthropathy is painful, invalidating and destructive. Authors report a prospective study of total knee arthroplasties in patients with severe haemophilia under continuous infusion of clotting factors. The purpose is to evaluate the benefits of continuous infusion of clotting factors regarding long-term functional improvement and radio-clinical results. METHODS: From 1998 to 2009, 20 total knee arthroplasties were implanted in 14 patients with a mean age of 36.5 years (24-56). A continuous infusion of anti-haemophilic factors was used and supervised by the physician of the Regional Haemophilia Treatment Centre (CRTH). Evaluation was clinical using the HSS and Oxford scores and radiological. RESULTS: One patient was lost to follow-up. Median follow-up is 66.5 months (6-134). Oxford score at latest follow-up is 42 (37-46). On revision, HSS score is 91 (84-96). Median flexion gain is 32.5° (-20; 75°). There is a median flexion contracture of 5° (0-15°) and a median extension improvement of 22.5°. We report 2 secondary infectious complications, concerning the same operated knee of a single patient. No post-operative haematoma was reported in our study. CONCLUSION: Total knee arthroplasty in haemophilic arthropathy improves both the function and quality of life of this group of patients. Continuous infusion of clotting factors contributes significantly to these results, by allowing early and intensive rehabilitation, and offers security regarding haemorrhagic complications commonly described in the literature and that we have not encountered in our study. LEVEL OF EVIDENCE: Therapeutic study, Level IV.

Diagnosis clinical criteria of sport related concussion: Toward an operational criteria definition in France.

OBJECTIVE: An expert working group was set up at the initiative of the French Ministry of Sports with the objective of harmonising the management of sport related concussion (SRC) in France, starting with its definition and diagnosis criteria. RESULTS: Definition: A clinical definition in 4 points have been established as follows: Concussion is a brain injury: 1) caused by a direct or indirect transmission of kinetic energy to the head; 2) resulting in an immediate and transient dysfunction of the brain characterised by at least one of the following disorders: a) Loss of consciousness, b) loss of memory, c) altered mental status, d) neurological signs; 3) possibly followed by one or more functional complaints (concussion syndrome); 4) the signs and symptoms are not explained by another cause. Diagnosis criteria: In the context of the direct or indirect transmission of kinetic energy to the head, the diagnosis of concussion may be asserted if at least one of the following signs or symptoms, observed or reported, is present within the first 24hours and not explained by another cause: 1) loss of consciousness; 2) convulsions, tonic posturing; 3) ataxia; 4) visual trouble; 5) neurological deficit; 6) confusion; 7) disorientation; 8) unusual behaviour; 9) amnesia; 10) headaches; 11) dizziness; 12) fatigue, low energy; 13) feeling slowed down, drowsiness; 14) nausea; 15) sensitivity to light/noise; 16) not feeling right, in a fog; 17) difficulty concentrating. CONCLUSION: Sharing the same definition and the same clinical diagnostic criteria for concussion is the prerequisite for common rules of management for all sports and should allow the pooling of results to improve our knowledge of this pathology.

Immunological crossreactivity between the human immunodeficiency virus type 1 virion infectivity factor and a 170-kD surface antigen of Schistosoma mansoni.

A monoclonal antibody (mAb) directed against a synthetic peptide derived from the sequence of the human immunodeficiency virus type 1 (HIV-1) regulatory protein virion infectivity factor (vif) labeled the surface of Schistosoma mansoni schistosomula by indirect immunofluorescence. Western blotting showed that two S. mansoni proteins of 170 and 65 kD were recognized by the mAb. Sera from 20% of S. mansoni-infected HIV-seronegative individuals tested recognized the PS4 peptide in an ELISA as did sera from S. mansoni-infected rats. Sera from individuals seropositive for HIV-1, but without schistosomiasis, that reacted with the vif peptide also recognized a 170-kD S. mansoni protein. This crossreactive S. mansoni antigen appears to be a target of immunity in vivo since passive transfer of the mAb VIF-CD3 to naive rats had a protective effect against a challenge infection with S. mansoni cercariae.

Expression of the alpha-1-antitrypsin gene in mononuclear phagocytes of normal and alpha-1-antitrypsin-deficient individuals.

To evaluate the contribution of mononuclear phagocytes, and particularly alveolar macrophages, to alpha-1-antitrypsin (alpha 1AT) production in normal and alpha 1AT-deficient individuals, Northern analysis with a human alpha 1AT complementary DNA was used to demonstrate that alpha 1AT messenger RNA (mRNA) can be detected in liver, blood monocytes, and alveolar macrophages. Quantification of alpha 1AT mRNA expression demonstrated that: (a) type PiMM monocytes and alveolar macrophages expressed, respectively, 200-fold and 70-fold less alpha 1AT mRNA per cell than the liver; (b) the level of expression of the alpha 1AT gene was increased during the in vitro maturation of blood monocytes; and (c) blood monocyte and alveolar macrophage levels of expression of the alpha 1AT gene were the same in PiMM and PiZZ individuals. However, the amount of newly synthesized alpha 1AT secreted by ZZ alveolar macrophages was 10 times lower than that secreted by MM alveolar macrophages. Thus, mononuclear phagocytes of PiZZ individuals express a secretory defect in alpha 1AT in a fashion similar to hepatocytes. Not only do mononuclear phagocytes provide a readily accessible cell to evaluate the regulation of alpha 1AT gene expression, but these cells may contribute to the levels of alpha 1AT present in the lower respiratory tract in the normal and ZZ states.

Evaluation of "at risk" alpha 1-antitrypsin genotype SZ with synthetic oligonucleotide gene probes.

Alpha 1-antitrypsin (alpha 1AT), a 52,000-mol-wt serum glycoprotein produced by hepatocytes and mononuclear phagocytes, functions as the major inhibitor of neutrophil elastase. The alpha 1AT haplotype S is associated with childhood liver disease and/or adult emphysema when inherited with the Z haplotype to give the phenotype SZ. To accurately identify the SZ phenotype at the level of genomic DNA, four 32P-labeled 19-mer synthetic oligonucleotide probes were prepared; two to identify the M and S difference in exon III, and two to identify the M and Z difference in exon V. These probes were hybridized with various cloned DNAs and genomic DNAs cut with the restriction endonucleases BgII and EcoRI; the genomic DNAs represented all six possible phenotype combinations of the M, S, and Z haplotypes (MM, MS, MZ, SS, ZZ, and SZ). Using the four probes to evaluate 42 samples of genomic DNA, the "at risk" SZ and ZZ phenotypes were correctly identified in all cases, as were the "not at risk" phenotypes SS, MS, MM, and MZ, demonstrating that both exon III and exon V directed probes are necessary to properly identify all of the major "at risk" alpha 1AT genes. However, when used to evaluate a very rare family carrying a null allele, these four oligonucleotide probes misidentified the "at risk" null-null and S null phenotypes as "not at risk" MM and SM combinations. These observations indicate that oligonucleotide gene probes yielded reliable and accurate assessment of "at risk" alpha 1AT genotypes in almost all situations, but in the context of prenatal diagnosis and genetic counseling this approach must be used with caution and in combination with family studies so as not to misidentify rare genotypes that may be associated with a risk for disease.

Carrier detection of Hemophilia B by using a restriction site polymorphism associated with the coagulation Factor IX gene.

The cloned complementary DNA for coagulation Factor IX (FIX) detects a frequent restriction fragment length polymorphism (RFLP) in human genomic DNAs digested with the restriction endonuclease Taq I. This genetic marker was used, in parallel with coagulation and immunological assays, to follow the segregation of an abnormal FIX gene in a large Hemophilia B family. Among the six potential female carriers, functional assays showed that four had a high probability, and two a low probability of being carriers. Analysis at the DNA level with the cDNA probe was informative in five of the six cases, and in all these five the diagnosis of carrier state was definitively confirmed. This demonstrates the feasibility of using linkage analysis at the DNA level for the genetic screening of Hemophilia B. This method has the advantages over conventional assays of giving a diagnosis of certainty, and of being applicable to early prenatal diagnosis using biopsies of trophoblast villi. At present, the single known polymorphism associated with the FIX gene restricts the application of linkage analysis to informative cases (40%), but findings of additional RFLPs in this region should improve this figure.

Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer.

A variety of pulmonary disorders, including cystic fibrosis, are potentially amenable to treatment in which a therapeutic gene is directly transferred to the bronchial epithelium. This is difficult to accomplish because the majority of airway epithelial cells replicate slowly and/or are terminally differentiated. Adenovirus vectors may circumvent this problem, since they do not require target cell proliferation to express exogenous genes. To evaluate the diversity of airway epithelial cell targets for in vivo adenovirus-directed gene transfer, a replication deficient recombinant adenovirus containing the Escherichia coli lacZ (beta-galactosidase [beta-gal]) gene (Ad.RSV beta gal) was used to infect lungs of cotton rats. In contrast to uninfected animals, intratracheal Ad.RSV beta gal administration resulted in beta-gal activity in lung lysate and cytochemical staining in all cell types forming the airway epithelium. The expression of the exogenous gene was dose-dependent, and the distribution of the beta-gal positive airway epithelial cells in Ad.RSV beta gal-infected animals was similar to the normal cell differential of the control animals. Thus, a replication deficient recombinant adenovirus can transfer an exogenous gene to all major categories of airway epithelial cells in vivo, suggesting that adenovirus vectors may be an efficient strategy for in vivo gene transfer in airway disorders such as cystic fibrosis.

Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.

[Secondary screening for osteoporosis in patients admitted for hip fracture to a rehabilitation center. Results of a survey]. / Dépistage de l'ostéoporose après fracture de hanche en milieu de rééducation. Résultats d'une enquête.

GOALS: To determine prevalence, risk factors and treatment of osteoporosis in patients with hip fracture observed in a rehabilitation ward. BACKGROUND: Hip fractures are associated with up to 20% excess mortality in the first year after fracture and cause functional disability in most survivors. Despite available risk indices and physician information, osteoporosis is still underdiagnosed and undertreated. METHOD: We obtained history, clinical and biological data, and bone density (BD) data in 41 patients admitted with hip fracture to a rehabilitation care centre. RESULTS: Only 3 patients had known osteoporosis. Although 50% had at least 1 clinical risk factor, all patients showed osteopenic BD scores and 68% had osteoporotic scores; only one was correctly treated. DISCUSSION: As with international studies, our study shows that osteoporosis is underdiagnosed. Risk assessment tools allow for routine screening and preventive measures incorporated into standard care practice. The prevention of osteoporotic fracture can be promoted in rehabilitation centres.

Crystallization and preliminary X-ray diffraction studies of a protective cloned 28 kDa glutathione S-transferase from Schistosoma mansoni.

Crystals of the recombinant 28 kDa glutathione S-transferase from Schistosoma mansoni have been obtained by the hanging-drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of this enzyme required the presence of a reducing agent and S-hexylglutathione. The crystals belong to the cubic space group P4(1)32 (or P4(3)32), with unit cell dimensions a = 122.6 A and contain one molecule in the asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray crystallographic structure analysis.

[Neurogenic muscle hypertrophy: imaging features in three cases and review of the literature]. / Hypertrophie musculaire neurogène: à propos de trois cas, imagerie et revue de la littérature.

OBJECTIVE: To review the literature on well-documented cases of neurogenic muscle hypertrophy in order to define significant features of this disease. PATIENTS AND METHODS: The PUBMED and SCIENCE DIRECT web-sites were used to conduct an inventory of all reported cases of this disease. We entered the key-words "hypertrophy", "muscle" and "neurogenic", and found 48 articles, describing 129 cases. Our criteria of inclusion included hypertrophy of one or several muscles of a lower limb, previous realization of at least one imaging study (CT or MRI) and electromyography of lower limbs; criterion of exclusion was hypertrophy related to hereditary or acquired polyneuropathies. Twenty-five cases were retained for investigation along with 3 recent cases observed in our department. RESULTS: Results show that neurogenic muscle hypertrophy is usually presents with painful enlargement of a calf in a male, aged 32 to 60 years, with previous history of low back pain and sciatica, 68% of the time due to disk herniation or lumbar stenosis. Other clinical findings may include radiation therapy or trauma. CONCLUSION: The symptoms of neurogenic muscle hypertrophy may lead to MRI examination before electromyography. This disease should be included in the differential diagnosis.

T helper cell epitopes of the human immunodeficiency virus (HIV-1) nef protein in rats and chimpanzees.

T helper cell antigenic and immunogenic determinants of the nef protein were investigated in the rat and chimpanzee models using recombinant nef protein and five synthetic peptides selected according to their amphipathic and alpha-helicity properties. The nef protein was shown to be immunogenic with both Freund's or aluminium hydroxide adjuvants. After immunization with the nef protein the 45-69 peptide was the most antigenic in rat and monkey models. In contrast, the 98-112 peptide, that required a carrier protein to induce in vitro rat T cell recall proliferation, was able to restimulate monkey T cells in the absence of a carrier. The amino acid sequence carrying the antigenic activity of the 45-69 peptide was further investigated by synthesizing short peptides overlapping this region. The antigenic sequence was precisely located in the middle of the peptide (region 50-59). This sequence was antigenic only when N alpha-acetylated. Circular dichroism analysis of the 45-69 peptide and the in vitro activity of the N-terminus group indicate in this case the involvement of the alpha-helical propensity for antigen presentation. However, the shorter sequence 50-64, able to induce a T cell reactivity, was determined as a beta-pleated sheet structure in aqueous solution. The 45-69 peptide was not only antigenic but also immunogenic and behaved in vivo as a functional T helper cell epitope. Indeed, the priming with the peptide or the transfer of peptide specific T cells to a naive recipient, followed by immunization with the nef protein, enhanced the subsequent antibody response to the nef protein. Together, these data indicate that the 45-69 peptide appears as a candidate for the in vivo elicitation of T cell immunity to the HIV-1 nef regulatory protein.

Determination of B-cell epitopes of nef HIV-I protein: immunogenicity related to their structure.

Determination of the B-cell epitopes of the nef molecule encoded by the human immunodeficiency virus type 1 (HIV-1) was undertaken using a set of six synthetic peptides. Sequences that were both antigenic and immunogenic and stimulated the production of antibodies recognizing the full length molecule, were considered as B-cell epitopes. Two peptidic sequences were antigenic both in rodents (mice and rats) and in non-human primates (chimpanzee). They were located in the regions 45-69 and 176-206 of the nef molecule. Two additional antigenic sequences were determined, one in chimpanzees, region 79-94, and the second in rodents, region 148-161. Immunogenicity was investigated in the rodents. Only the 45-69 and 176-206 sequences were immunogenic, and specific antibodies present in the sera of the immunized animals reacted with the nef protein. Therefore, each of these two sequences could be considered as containing at least one B-cell epitope. The fine epitopic specificity was determined using subfragments of these two sequences and it was shown that the antigenic determinants were contained in the C-terminal region of each sequence overlapping with the T-cell epitopes. These results raised the importance of vicinity of B- and T-cell determinants and their immunogenicity.

[The accessory soleus muscle: a report of 21 cases and a review of the literature]. / Le muscle soléaire accessoire: à propos de 21 observations et revue de la littérature.

PURPOSE OF THE STUDY: Well known to anatomy specialists, the accessory soleus muscle was first demonstrated to be involved in painful syndromes in 1965 (Dunn). This supranumerary muscle situated in front of the calcaneum can be taken for a soft tissue tumor. The purpose of this work was to report a series of 21 patients with an accessory soleus muscle and to present the characteristic features, diagnostic methods, and treatment indications and modalities. MATERIAL AND METHODS: This series included 20 patients (one symptomatic bilateral case), fourteen men and six women, mean age 25 years. Seventeen patients practiced sports and ten had had a prior operation. All patients complained of exercise-related pain. The physical examination was normal with the exception of a tumefaction, which was soft at rest and hard at triceps contraction against resistance, lying laterally to the Achilles tendon. We studied plain x-rays, ultrasound studies, computed tomographies, and electromyograms and later MRI which became the reference method to demonstrate the details of normal muscle structure. Ten patients (one bilateral case) were not particularly bothered by the supernumerary muscle. Functional treatment was given and provided patient satisfaction. For the other ten patients, who wished to continue their physical activities, two underwent fasciotomy (including our first case where fasciotomy was undertaken because a tumor was suspected) and eight underwent resection of the supranumerary muscle. RESULTS: The patients were followed for 6 to 19 years. Outcome was very good in all patients who were free of pain and had complete joint movement with symmetrical muscle force. Normal sports activities were resumed. DISCUSSION: The accessory soleus muscle is found in 10% of individuals. It is often asymptomatic. The muscle inserts on the anterior aspect of the soleus muscle or on the posterior aspect of the tibia or the muscles of the deep posterior compartment. It lies anterior to the calcaneal tendon and terminates on the calcaneal tendon or the superior or medial aspect of the calcaneus via fleshy fibers or a distinct tendon. Frequent in primates, this anatomic variant is present during embryological development. Its persistence depends on phylogenetic evolution. Among other hypotheses (exercise-induced intermittent claudication, compression of the tibial nerve, excessive tension on the nerve innervating the accessory soleus muscle), this supranumerary muscle is generally considered to be the cause of a localized compartment syndrome. Pain experienced during exercise is the only symptom regularly reported by patients. A careful examination is required to rule out another local cause. Besides tumefaction lateral to the Achilles tendon, often found bilaterally, there is no other clinical sign. Plain x-rays, ultrasound and computed tomography simply demonstrate a "mass" in front of the Achilles tendon. MRI is the examination of choice enabling confirmation of the muscle nature of the mass and ruling out the possible diagnosis of tumor. Since there is no risk of aggravation, surgical treatment can be avoided if there is no complaint. If the patient is seriously impaired, surgery can be proposed. In our opinion, complete resection of the supernumerary muscle is the safest solution and should be preferred over simple fasciotomy.

New versatile cloning and sequencing vectors based on bacteriophage M13.

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.

Fusion of restriction termini using synthetic adaptor oligonucleotides.

A method is proposed for linking a DNA fragment possessing a 5' single-stranded extension to one carrying a 3' extension. Synthetic oligonucleotide adaptors can be used to (i) change the site specificity at the termini of a fragment generated by restriction enzyme cleavage and (ii) simultaneously dephosphorylate the extremities of a DNA molecule to prevent recircularisation and allow positive selection for recombinant DNA molecules.

cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2+ mobilization.

The serine protease alpha-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exosite of thrombin.