Clinical relevance of nitrated beta 2-glycoprotein I in antiphospholipid syndrome: Implications for thrombosis risk.

Β2-Glycoprotein I (ß2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of ß2GPI was examined.The effects of nitrated (n)ß2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. ß2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. ß2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated ß2GPI loses this binding (p < 0.05) and had diminished activity in inhibiting platelet adhesion to vWF under high shear flow (p < 0.01). Levels of nß2GPI were increased in patients with primary APS compared to patients with either secondary APS (p < 0.05), autoimmune disease without APS (p < 0.05) or non-autoimmune patients with arterial thrombosis (p < 0.01) and healthy individuals (p < 0.05).In conclusion tyrosine nitration of plasma ß2GPI is demonstrated and has important implications with regards to the pathophysiology of platelet mediated thrombosis in APS. Elevated plasma levels of nß2GPI in primary APS may be a risk factor for thrombosis warranting further investigation.

Quantitation of Total and Free Thiol ß2-Glycoprotein I Levels for Diagnostic and Prognostic Purposes in the Antiphospholipid Syndrome.

ß2-Glycoprotein I is the major autoantigen in the antiphospholipid syndrome (APS), a prothrombotic disorder characterized by the occurrence of either venous or arterial thrombosis. In women it is also associated with an increased risk of obstetric complications such as recurrent miscarriages. We have identified that the plasma protein ß2-glycoprotein I in healthy individuals exists in an optimal ratio between two distinct forms, an oxidized and free thiol, reduced form. This ratio is disrupted in pathophysiological conditions associated with increased oxidative stress such as the APS, but also in the setting of age-related macular degeneration and gram-negative sepsis. We have developed assays that quantify plasma/serum levels of total and free thiol ß2-glycoprotein I which can potentially be used for risk stratification and prognostic purposes in the early stages of the aforementioned conditions.

Quantitation of Total and Free Thiol Angiotensinogen as a Prognostic Marker for Preeclampsia.

Angiotensinogen mediates an important role in the pathophysiology of preeclampsia, a disorder of pregnancy characterized by hypertension and proteinuria usually after 20 weeks of gestation. Angiotensinogen is found in two distinct posttranslational forms in the plasma, an oxidized and a reduced (free thiol) form. Higher levels of the oxidized form are associated with an increased risk of preeclampsia. We have developed novel ELISA assays to quantitate the levels of total and free thiol angiotensinogen allowing for calculation of the amount of oxidized angiotensinogen species. We describe the methodology for performing these assays.

Molecular pathophysiology of the antiphospholipid syndrome: the role of oxidative post-translational modification of beta 2 glycoprotein I.

It has been well established that antiphospholipid antibodies and specifically those directed against beta 2 glycoprotein I (ß2GPI) are pathogenic for the development of thrombosis in the antiphospholipid syndrome (APS). Several groups have shown that anti-ß2GPI antibodies, in complex with ß2GPI, elicit effects on blood cells and coagulation-fibrinolysis proteins, which prime the arterial and venous vasculature for the development of thrombosis. However, much less is known about the mechanism initiating the production of autoantibodies against ß2GPI, a physiological abundant protein of blood. In the current review, novel findings are presented regarding the structure and oxidative post-translational modifications of ß2GPI, which trigger the immune response. The majority of circulating ß2GPI exists in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of ß2GPI. The free thiols exposed on ß2GPI are involved in the interaction with platelets and endothelial cells. We propose that this abundant pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules from oxidative stress. Oxidation of ß2GPI confers an increase in its immunogenicity through a Th1 immunological mechanism. The clinical significance of these observations is that serum from patients with APS, assessed by a novel ELISA assay, have a significant increase in oxidised ß2GPI. These findings hold promise, not only for the delineation of the role of ß2GPI as an immunological target, but also for the development of improved diagnostic and prognostic assays for APS.

Redox control of ß2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

BACKGROUND: ß(2) -Glycoprotein I (ß(2) GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of ß(2) GPI in thrombus formation is unknown. We have recently shown that ß(2) GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin-1 and protein disulfide isomerase, and that reduction of ß(2) GPI can take place on the platelet surface. METHODS: ß(2) GPI, reduced by thioredoxin-1, was labeled with the selective sulfhydryl probe N(a)-(3-maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced ß(2) GPI for von Willebrand factor (VWF) and the effect of reduced ß2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin-activated VWF was studied in the presence of reduced ß(2) GPI. RESULTS: We demonstrate that the Cys288-Cys326 disulfide in domain V of ß(2) GPI is the predominant disulfide reduced by thioredoxin-1. Reduced ß(2) GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. ß(2) GPI reduced by thioredoxin-1, in comparison with non-reduced ß(2) GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. CONCLUSIONS: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol-dependent interaction of ß(2) GPI with VWF may contribute to the redox regulation of platelet adhesion.

In vivo modulation of angiogenesis by beta 2 glycoprotein I.

Beta 2 glycoprotein I (ß2GPI) is the major auto antigen in the antiphospholipid syndrome but also interacts with fibrinolytic and angiogenic proteins. The aim of this study was to examine the angiogenic potential of ß2GPI in vivo in ß2GPI deficient mice utilizing angiogenic assays. ß2GPI deficient mice show increased microvessel formation in comparison to ß2GPI replete controls when injected with growth factor free-matrigel implants. However, microvessel formation in matrigel plugs of ß2GPI deficient mice was less than in ß2GPI replete mice when basic fibroblast growth factor (bFGF) was included in the matrigel. Hemoglobin content was higher in vascular endothelial growth factor (VEGF) containing-matrigel plugs in the ß2GPI deficient mouse demonstrating that the lack of ß2GPI led to increased extravasation by VEGF. Melanoma B16F10 tumour growth was enhanced in ß2GPI deficient mice. Melanoma microvessel density was increased in ß2GPI deficient mice but the proliferation rate of tumour cells (determined by Ki67 immunohistochemistry) was unaffected by the presence or absence of ß2GPI. Subcutaneous delivery of native human ß2GPI by the ALZET osmotic pump did not affect melanoma tumour growth in ß2GPI deficient mice. We conclude that the in vivo unopposed action of ß2GPI is anti-angiogenic however this function is modified in the presence of a strong angiogenic stimulus into stabilization of vessel formation. Although the presence of ß2GPI attenuates vessel sprouting in certain tumours, no survival benefit is conferred to tumour bearing animals. This does not preclude the potential benefit of modified or fragments of ß2GPI in anti-angiogenesis research.

Risk stratification and outcome of cellulitis admitted to hospital.

OBJECTIVES: To identify risk factors associated with mortality and adverse outcome of community acquired cellulitis/erysipelas requiring hospital admission. METHODS: A retrospective analysis of 395 episodes of cellulitis/erysipelas admitted to a tertiary referral hospital between January 1999 and December 2006. RESULTS: Mortality was 2.5% (10/395). There were 112 complications (28.4%). Median hospitalisation was 5 days. Factors independently associated with mortality, adverse outcome and prolonged stay (>7 days) were bacteraemia and albumin <30 g/L. A risk stratification model was designed based on factors independently associated with adverse outcome: altered mental status, neutrophilia/paenia, discharge from the cellulitic area, hypoalbuminaemia and history of congestive cardiac failure. Adverse outcome risk among patients with scores <4, 6-9 and >9 was <20%, 55% and 100%, respectively. All patients who died had admission score >or=4. Factors independently associated with prolonged hospitalisation were: age >60, symptom duration >4 days, hypoalbuminaemia, bacteraemia, isolation of MRSA and time to effective antibiotics >8 h. MRSA was more frequent among patients admitted during 2003-2006 (OR 2.43, 95% CI: 1-12-5.27). Streptococci accounted for most bacteraemia (11/20). Infectious Disease physician input was independently associated with shorter hospitalisation. CONCLUSIONS: Cellulitis/erysipelas requiring hospitalisation confers considerable morbidity and mortality. Clinical markers present on admission can be used to stratify patient risk of mortality and adverse outcome.

Beta2-glycoprotein I inhibits vascular endothelial growth factor and basic fibroblast growth factor induced angiogenesis through its amino terminal domain.

BACKGROUND: Beta-2 glycoprotein I (beta(2)GPI) is a plasma glycoprotein which interacts with various proteins of the coagulation and fibrinolysis system. beta(2)GPI has recently been shown to have anti-angiogenic properties. OBJECTIVES: We undertook this study to investigate the specific domain of beta(2)GPI involved in the anti-angiogenic function and its effect on downstream signaling of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). METHODS: Various preparations of beta(2)GPI were used on human umbilical vein endothelial cells (HUVECs) in the absence or presence of VEGF and bFGF. The effect on HUVECs' proliferation, migration and tubule formation in Matrigel matrix was investigated. The effect of beta(2)GPI on the mRNA expression of VEGF receptors and phosphorylation of signaling molecules was also studied. RESULTS: beta(2)GPI is shown in this study to be an anti-angiogenic molecule in vitro by inhibiting VEGF and bFGF-induced proliferation, migration and papillary-like tubule formation of HUVECs. This inhibition was achieved by native, proteolytically clipped and domain deletion mutants, domain I-IV (DI-IV) but not domain II-V (DII-V) of beta(2)GPI. Native beta(2)GPI was found to downregulate the expression of the VEGF receptor KDR/Flk-1 on endothelial cells and to block the phosphorylation of VEGF's downstream effector molecules in the MAPK/ERK and PI3K/Akt/GSK3beta pathways. CONCLUSIONS: These results indicate that beta(2)GPI has anti-angiogenic functions which depend on the presence of domain I. This anti-angiogenic activity may have important implications for the therapeutic manipulation of angiogenesis in various disease states.

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).

New clinical, laboratory and experimental insights, since the 1999 publication of the Sapporo preliminary classification criteria for antiphospholipid syndrome (APS), had been addressed at a workshop in Sydney, Australia, before the Eleventh International Congress on antiphospholipid antibodies. In this document, we appraise the existing evidence on clinical and laboratory features of APS addressed during the forum. Based on this, we propose amendments to the Sapporo criteria. We also provide definitions on features of APS that were not included in the updated criteria.

Pathogenic role of anti-beta 2-glycoprotein I antibodies in antiphospholipid associated fetal loss: characterisation of beta 2-glycoprotein I binding to trophoblast cells and functional effects of anti-beta 2-glycoprotein I antibodies in vitro.

BACKGROUND: Antiphospholipid antibodies reacting with beta2-glycoprotein I (beta 2GPI) have been associated with recurrent fetal loss and pregnancy complications. OBJECTIVE: To investigate whether specific mutations in the phospholipid binding site of beta 2GPI might affect its binding to trophoblast and in turn the anti-beta 2GPI antibody induced functional effects. METHODS: beta 2GPI adhesion to trophoblast was evaluated as human monoclonal IgM or polyclonal IgG anti-beta 2GPI antibody binding to trophoblast monolayers cultured (1) in complete medium; (2) in serum-free medium; (3) after serum starvation in the presence of purified human beta 2GPI; or (4) in the presence of beta 2GPI with single or multiple mutations in the amino acid loop Cys(281)-Lys-Asn-Lys-Glu-Lys-Lys-Cys(288). The effect of anti-beta 2GPI binding to trophoblast was evaluated as chorionic gonadotropin (hCG) mRNA expression, and protein release by RT-PCR and radioimmunoassay, respectively. RESULTS: beta 2GPI adhesion to trophoblast and its consequent recognition by the specific antibodies were inversely proportional to the mutation number in the phospholipid binding site. Anti-beta 2GPI antibodies reduced gonadotropin release, hormone dependent hCG mRNA expression, and protein synthesis in the presence of beta 2GPI, while the addition of the mutants or the absence of beta 2GPI had no effect. CONCLUSIONS: beta 2GPI binds to trophoblast in vitro through its fifth domain, as reported for endothelial cells, and can be recognised by anti-beta 2GPI antibodies; the antibody binding downregulates trophoblast hCG synthesis and secretion. Such a mechanism might contribute to defective placentation in women with fetal loss associated with the antiphospholipid syndrome.

Anti-beta2-glycoprotein I autoantibodies require an antigen density threshold, consistent with divalent binding.

Autoantibodies binding beta2-glycoprotein I (B2GPI) are an important finding in the antiphospholipid syndrome. While antibodies from mice or rabbits immunized with B2GPI readily bind B2GPI coated on a polystyrene microwell plate, anti-B2GPI autoantibodies only do so when using a modified microwell plate with a negatively charged surface. This study demonstrates that, for the detection of anti-B2GPI autoantibodies in an ELISA using modified plates, an antigen coating concentration threshold exists, such that minimal or no binding occurs below a certain coating concentration of antigen, even though antigen is easily demonstrable on the plate. This is consistent with the hypothesis that autoantibodies require divalent binding to B2GPI for detection, as sufficient antigen density for two protein molecules to be sufficiently close to enable divalent binding would only be expected to occur at higher coating concentrations. Several mutant forms of B2GPI developed for epitope determination experiments are shown to have decreased binding to microtitre plates compared to wild-type. If wild-type and mutants are assayed for antibody binding near the threshold a significant diminution in binding to mutants occurs that is the result of inadequate binding to the plate, but could be misinterpreted as the result of interruption of an epitope by the mutation.

Distinguishing features of anti-beta2 glycoprotein I antibodies between patients with leprosy and the antiphospholipid syndrome.

Anticardiolipin (ACA), anti-beta2 glycoprotein I (beta2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10(-2) to 10(-3)). Levels of IgM anti-beta2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both beta2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-beta2GPI antibody levels. Likewise, a large fraction of anti-beta2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-beta2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine beta2GPI immobilized on non-irradiated polystyrene plates, (ii) concentration-dependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase beta2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the beta2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain I- and V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-beta2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.

Human tryptase epsilon (PRSS22), a new member of the chromosome 16p13.3 family of human serine proteases expressed in airway epithelial cells.

Probing of the GenBank expressed sequence tag (EST) data base with varied human tryptase cDNAs identified two truncated ESTs that subsequently were found to encode overlapping portions of a novel human serine protease (designated tryptase epsilon or protease, serine S1 family member 22 (PRSS22)). The tryptase epsilon gene resides on chromosome 16p13.3 within a 2.5-Mb complex of serine protease genes. Although at least 7 of the 14 genes in this complex encode enzymatically active proteases, only one tryptase epsilon-like gene was identified. The trachea and esophagus were found to contain the highest steady-state levels of the tryptase epsilon transcript in adult humans. Although the tryptase epsilon transcript was scarce in adult human lung, it was present in abundance in fetal lung. Thus, the tryptase epsilon gene is expressed in the airways in a developmentally regulated manner that is different from that of other human tryptase genes. At the cellular level, tryptase epsilon is a major product of normal pulmonary epithelial cells, as well as varied transformed epithelial cell lines. Enzymatically active tryptase epsilon is also constitutively secreted from these cells. The amino acid sequence of human tryptase epsilon is 38-44% identical to those of human tryptase alpha, tryptase beta I, tryptase beta II, tryptase beta III, transmembrane tryptase/tryptase gamma, marapsin, and Esp-1/testisin. Nevertheless, comparative protein structure modeling and functional studies using recombinant material revealed that tryptase epsilon has a substrate preference distinct from that of its other family members. These data indicate that the products of the chromosome 16p13.3 complex of tryptase genes evolved to carry out varied functions in humans.

Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin.

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta(2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0.05) and at lower antibody concentrations (12.5 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(2)GPI (r = 0.90; P < 0.001) but not to CL (r = 0.06; P = 0.76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.

Mast cells/basophils in the peripheral blood of allergic individuals who are HIV-1 susceptible due to their surface expression of CD4 and the chemokine receptors CCR3, CCR5, and CXCR4.

A population of metachromatic cells with mast cell (MC) and basophil features was identified recently in the peripheral blood of patients with several allergic disorders. This study now shows that these metachromatic cells express on their surface the high-affinity IgE receptor (FcepsilonRI), CD4, and the chemokine receptors CCR3, CCR5, and CXCR4, but not the T-cell surface protein CD3 and the monocyte/macrophage surface protein CD68. This population of MCs/basophils can be maintained ex vivo for at least 2 weeks, and a comparable population of cells can be generated in vitro from nongranulated hematopoietic CD3(-)/CD4(+)/CD117(-) progenitors. Both populations of MCs/basophils are susceptible to an M-tropic strain of human immunodeficiency virus 1 (HIV-1). Finally, many patients with acquired immunodeficiency syndrome have HIV-1-infected MCs/basophils in their peripheral blood. Although it is well known that HIV-1 can infect CD4(+) T cells and monocytes, this finding is the first example of a human MC or basophil shown to be susceptible to the retrovirus. (Blood. 2001;97:3484-3490)

Tryptase 4, a new member of the chromosome 17 family of mouse serine proteases.

Genomic blot analysis raised the possibility that uncharacterized tryptase genes reside on chromosome 17 at the complex containing the three genes that encode mouse mast cell protease (mMCP) 6, mMCP-7, and transmembrane tryptase (mTMT). Probing of GenBank's expressed sequence tag data base with these three tryptase cDNAs resulted in the identification of an expressed sequence tag that encodes a portion of a novel mouse serine protease (now designated mouse tryptase 4 (mT4) because it is the fourth member of this family). 5'- and 3'-rapid amplification of cDNA ends approaches were carried out to deduce the nucleotide sequence of the full-length mT4 transcript. This information was then used to clone its approximately 5.0-kilobase pair gene. Chromosome mapping analysis of its gene, sequence analysis of its transcript, and comparative protein structure modeling of its translated product revealed that mT4 is a new member of the chromosome 17 family of mouse tryptases. mT4 is 40-44% identical to mMCP-6, mMCP-7, and mTMT, and this new serine protease has all of the structural features of a functional tryptase. Moreover, mT4 is enzymatically active when expressed in insect cells. Due to its 17-mer hydrophobic domain at its C terminus, mT4 is a membrane-anchored tryptase more analogous to mTMT than the other members of its family. As assessed by RNA blot, reverse transcriptase-polymerase chain reaction, and/or in situ hybridization analysis, mT4 is expressed in interleukin-5-dependent mouse eosinophils, as well as in ovaries and testes. The observation that recombinant mT4 is preferentially retained in the endoplasmic reticulum of transiently transfected COS-7 cells suggests a convertase-like role for this integral membrane serine protease.

Impaired thrombin generation in beta 2-glycoprotein I null mice.

Autoimmune antibodies to beta(2)-glycoprotein I (beta2GPI) have been proposed to be clinically relevant because of their strong association with thrombosis, miscarriage, and thrombocytopenia. By using a homologous recombination approach, beta2GPI-null mice were generated to begin to understand the physiologic and pathologic role of this prominent plasma protein in mammals. When beta2GPI heterozygotes on a 129/Sv/C57BL/6 mixed genetic background were intercrossed, only 8.9% of the resulting 336 offspring possessed both disrupted alleles. These data suggest that beta2GPI plays a beneficial role in implantation and/or fetal development in at least some mouse strains. Although those beta2GPI-null mice that were born appeared to be relatively normal anatomically and histologically, subsequent analysis revealed that they possessed an impaired in vitro ability to generate thrombin relative to wild type mice. Thus, beta2GPI also appears to play an important role in thrombin-mediated coagulation.

Epitope studies with anti-beta 2-glycoprotein I antibodies from autoantibody and immunized sources.

This paper examines the methodology of anti-beta(2)-glycoprotein I (beta(2)-GPI) epitope determination and provides further epitope studies using human sera containing anti-beta(2)-GPI autoantibodies. Studies in this field may be misleading as the antigen coating density using mutant forms of beta(2)-GPI may be below the threshold required for monogamous divalent binding by low affinity anti-beta(2)-GPI autoantibodies, while being easily detected by high affinity anti-beta(2)-GPI from immunized animals. The antigen density threshold effect is found in anti-beta(2)-GPI autoantibodies from humans and from monoclonal anti-beta(2)-GPI derived from mice with models of autoimmune disease. Anti-beta(2)-GPI from an autoimmune mouse and from 18/21 human sera did not bind above background levels to a domain-I-deleted mutant. In addition, single point mutations in domain I result in dramatic changes in the binding of many human sera containing anti-beta(2)-GPI. These findings support a conclusion that domain I of beta(2)-GPI contains significant epitopes for the anti-beta(2)-GPI antibodies found in the antiphospholipid syndrome.

Identification of a new member of the tryptase family of mouse and human mast cell proteases which possesses a novel COOH-terminal hydrophobic extension.

Mapping of the tryptase locus on chromosome 17 revealed a novel gene 2.3 kilobase 3' of the mouse mast cell protease (mMCP) 6 gene. This 3.7-kilobase gene encodes the first example of a protease in the tryptase family that contains a membrane-spanning segment located at its COOH terminus. Comparative structural studies indicated that the putative transmembrane tryptase (TMT) possesses a unique substrate-binding cleft. As assessed by RNA blot analyses, mTMT is expressed in mice in both strain- and tissue-dependent manners. Thus, different transcriptional and/or post-transcriptional mechanisms are used to control the expression of mTMT in vivo. Analysis of the corresponding tryptase locus in the human genome resulted in the isolation and characterization of the hTMT gene. The hTMT transcript is expressed in numerous tissues and is also translated. Analysis of the tryptase family of genes in mice and humans now indicates that a primordial serine protease gene duplicated early and often during the evolution of mammals to generate a panel of homologous tryptases in each species that differ in their tissue expression, substrate specificities, and physical properties.