Effect of tissue transglutaminase on the solubility of proteins containing expanded polyglutamine repeats.

The expansion of a polyglutamine (polyQ) domain in neuronal proteins is the molecular genetic cause of at least eight neurodegenerative diseases. Proteins with a polyQ domain that is greater than 40 Q (Q40) residues form insoluble intranuclear and cytoplasmic inclusions. Expanded polyQ proteins self-associate by non-covalent interactions and become insoluble. They can also be covalently cross-linked by tissue transglutaminase (TTG), a calcium-dependent enzyme present in cells throughout the nervous system. However, it remains unclear whether TTG cross-linking directly contributes to the insolubility of the expanded polyQ proteins. Using an in vitro solubility assay, we found TTG cross-linked Q62 monomers into high molecular weight soluble complexes in a calcium-dependent reaction. Inhibition of TTG cross-linking by primary amine substrates including putrescine and biotinylated pentylamine antagonized TTG's ability to form soluble complexes. In contrast, primary amines (histamine and lysine) that were less effective inhibitors of TTG cross-linking did not inhibit Q62 from becoming insoluble. In summary, TTG can increase the solubility of expanded polyQ proteins by catalyzing intermolecular cross-links. This demonstrates directly that TTG will reduce the ability of expanded polyQ proteins from becoming insoluble. Furthermore, the effectiveness of a primary amine substrate at inhibiting formation of insoluble inclusions may be related to their ability to inhibit intermolecular cross-linking by TTG.

Reversible shear-mediated platelet dysfunction during cardiac surgery as assessed by the PFA-100 platelet function analyzer.

We undertook this investigation to assess alterations in shear-mediated platelet function during cardiac surgery and to determine the potential for the PFA-100 to predict post-operative bleeding. Platelet aggregation and PFA-100 closure times were determined in 18 adult patients at five intervals during cardiac surgery. Associations between post-operative bleeding and closure times were examined in an additional 58 patients. Statistical analysis consisted of Student's t, Wilcoxon signed rank, and Spearman correlation tests. All results are reported as mean +/- SEM. Collagen/epinephrine closure times were prolonged prior to and throughout surgery. Collagen/adenosine-5'-diphosphate (ADP) closure times were significantly prolonged by heparin administration, 141 +/- 15 s versus 115 +/- 10 s (P = 0.01), and subsequent initiation of cardiopulmonary bypass (CPB), 203 +/- 12 s (P= 0.0001); however, 15 min after protamine administration, closure times returned to near pre-operative values, 138 +/- 12 s (P = not significant). In contrast, platelet aggregation in response to ADP remained impaired in 17 of 19 patients after CPB. Neither ex vivo correction of sample hematocrits nor supplementation with Humate P affected closure times. Positive and negative predictive values for post-CPB collagen/ADP closure times to predict bleeding were 18 and 96%, respectively. These results suggest that factors both intrinsic and extrinsic to the platelet contribute to reversible shear-mediated platelet dysfunction during CPB, and that the PFA-100 may prove useful after CPB to identify patients unlikely to benefit from platelet transfusions.

Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase.

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.

Hemostatic effects of antithrombin III supplementation during cardiac surgery: results of a prospective randomized investigation.

Failure to suppress thrombin generation during cardiac surgery promotes fibrin generation, fibrinolysis, and a consumptive coagulopathy. Acquired deficiencies of antithrombin III may play a contributory role. We hypothesized that antithrombin III supplementation to normal physiologic concentrations would decrease thrombin generation and potentially reduce peri-operative bleeding. Twenty patients undergoing coronary artery bypass graft surgery were randomized for this prospective, double-blind, placebo-controlled study. Ten patients received antithrombin III supplementation (50 U/kg) by intravenous infusion prior to incision, and 10 patients received a placebo. Blood samples were obtained pre-operatively, at 1 and 2 h following initiation of cardiopulmonary bypass (CPB), and at 1, 3, and 24 h after completion of CPB. Samples were analyzed for antithrombin III, thrombin-antithrombin III (TAT) complex, and D-dimer concentrations. Cumulative blood loss was recorded at 6 and 12 h after CPB. No statistically significant differences in patient demographics or total heparin dose administered were observed between groups. As expected, plasma antithrombin III concentrations were maintained near pre-operative values in the treatment group, but not in the placebo group. Despite this difference, no statistically significant alterations in generation of TAT complex, D-dimer, or blood loss occurred between groups. Antithrombin III supplementation to maintain normal physiologic concentrations during CPB did not alter significantly thrombin generation, fibrinolytic activity, or blood loss in adults undergoing elective cardiac surgery.

Localization of tissue transglutaminase in human carotid and coronary artery atherosclerosis: implications for plaque stability and progression.

Although atherosclerosis progresses in an indolent state for decades, the rupture of plaques creates acute ischemic syndromes that may culminate in myocardial infarction and stroke. Mechanical forces and matrix metalloproteinase activity initiate plaque rupture, whereas tissue inhibitors of metalloproteinases have an important (albeit indirect) role in plaque stabilization. In this paper, an enzyme that could directly stabilize the plaque is described. Tissue transglutaminase (TG) catalyzes the formation of epsilon(gamma-glutamyl)lysine isopeptide bonds that are resistant to enzymatic, mechanical, and chemical degradation. We performed immunohistochemistry for TG in atherosclerotic human coronary and carotid arteries. TG was most prominent along the luminal endothelium and in the medium of the vessels with a distribution mirroring that of smooth muscle cells. Variable, often prominent, immunoreactivity for TG was also seen in the intima, especially in regions with significant neovascularization. Additionally, TG was detected in fibrous caps and near the "shoulder regions" of some plaques. A monoclonal antibody to the transglutaminase product epsilon(gamma-glutamyl)lysine isopeptide demonstrated co-localization with TG antigen. Transglutaminase activity was found in 6 of 14 coronary artery atherectomy samples. Cross-linking of TG substrates such as fibrinogen, fibronectin, vitronectin, collagen type I, and protease inhibitors stabilized the plaque. Furthermore, the activation of transforming growth factor-beta-1 by TG might be an additional mechanism for the promotion of plaque stabilization and progression by increasing the synthesis of extracellular matrix components.

Tamoxifen inhibits angiogenesis in estrogen receptor-negative animal models.

Inhibition of tumor angiogenesis is a therapeutic strategy that can inhibit tumor growth and metastases. The aim of this study was to determine whether the estrogen receptor (ER) ligand drug tamoxifen has antiangiogenic effects. We used three different models of angiogenesis, including measurement of microvessel densities in murine tumors, ex vivo aortic ring assays, and corneal pocket assays. ER-negative fibrosarcoma tumors in tamoxifen-treated ovariectomized rats had significantly less vessel formation compared with untreated animals (median microvessel density, 53.6 versus 94.3 counts/per x 200 field; P = 0.002). Rat aortic rings treated with tamoxifen at several different concentrations demonstrated significantly less vascular sprouting than control rings (P = 0.0001). Corneal pocket assays performed in tamoxifen-treated rats compared with control and estrogen-treated rats demonstrated decreased vascular length (0.88 mm versus 1.26 mm versus 1.47 mm; P = 0.022) and vessel area (21% versus 34% versus 47%; P = 0.018). These three animal models all showed significant inhibition of angiogenesis by tamoxifen and suggest a possible contributory mechanism of ER-independent manipulation by tamoxifen in the treatment and prevention of breast cancer. These studies raise the question as to whether or not newer ER ligand drugs might possess even more potent antiangiogenic effects, which in turn could lead to the broadening of the clinical usefulness of these compounds in a number of diseases. More importantly, these studies suggest that the antiangiogenic effects of tamoxifen are due, in part, to ER-independent mechanisms.

Assessment of primary hemostasis by PFA-100 analysis in a tertiary care center.

We evaluated the utility of the PFA-100 platelet function analyzer in identifying disorders in platelet function and/or von Willebrand factor (vWF) in patients with various systemic disorders being followed at a tertiary care center. Closure times were determined with collagen/ ADP (CADP) and collagen/epinephrine (CEPI) cartridges for 305 patients, and abnormal results were further evaluated with platelet aggregometry and vWF analysis. Prolonged CADP and/or CEPI closure times were identified in 114 patients (37.3%), but most were isolated prolonged CEPI closure times predominantly due to aspirin therapy (79 patients). Prolonged CADP closure times were most frequently due to qualitative platelet defects and/or decreased vWF levels. Prolonged CADP closure times were encountered most frequently in patients with sickle cell disease and were associated with a decreased hematocrit. This study demonstrated that the PFA-100 analyzer can accurately assess vWF-dependent platelet function and detect other platelet defects under high shear stress in complex patient populations.

Pain assessment in infants and children.

The science of pain assessment for infants and children has grown substantially in the past several decades to the point that valid and reliable methods for pain assessment are available for use in clinical settings. Accurate pain assessment requires consideration of children's developmental level, type of pain experienced, history and context of pain, family influences, and interaction with the health care team. Research is needed to improve the sensitivity, specificity, and generalizability of pain-assessment tools and to more fully incorporate contextual factors into the objective assessment process. Finally, the improvement of pain assessment in the clinical setting can be viewed as a patient care quality issue, and continuous quality improvement methods can be used effectively to incorporate pain assessment as an integral component of every infant's and child's health care.

Myocardial ischemia correlates with reduced fibrinolytic activity following peripheral vascular surgery.

STUDY OBJECTIVES: To evaluate the relationship between perioperative ischemia and serial concentrations of D-dimer, which is a sensitive and specific marker of fibrinolytic activity. Myocardial ischemia and infarction are well-recognized complications of peripheral vascular surgery. We hypothesized that patients at increased risk of perioperative myocardial ischemia might be identified preoperatively by abnormal hemostatic indices. DESIGN: Prospective clinical outcomes study. SETTING: A 1,124-bed tertiary care medical center. PATIENTS: 42 ASA physical status II, III, and IV patients undergoing peripheral vascular surgery. INTERVENTIONS: Serial D-dimer concentrations were measured preoperatively, and at 24 and 72 hours postoperatively. Continuous 12-lead ST-segment monitoring (Mortara Instrument, Inc., Milwaukee, WI) was performed with the acquisition of a 12-lead ECG every 20 seconds for 72 hours. MEASUREMENTS AND MAIN RESULTS: D-dimer measurements were performed in duplicate using the Dimer Gold assay (American Diagnostica, Greenwich CT). Ischemic episodes, as defined by continuous 12-lead ST-segment monitoring, occurred in 49% of patients. There were no demographic differences between ischemic and nonischemic groups. Although baseline D-dimer concentrations were not statistically significantly different between groups, patients experiencing perioperative myocardial ischemia generated significantly less D-dimer during the perioperative period (p = 0. 014). CONCLUSIONS: PATIENTS with an impaired fibrinolytic response, as defined by reduced generation of D-dimer, experienced an increased incidence of perioperative myocardial ischemia.

Early wound healing exhibits cytokine surge without evidence of hypoxia.

OBJECTIVE: To ascertain the spatial and temporal relation of wound hypoxia to the cell types involved, expression of selected angiogenic cytokines, the proliferative status of cells in the wound site, and angiogenesis. SUMMARY BACKGROUND DATA: Hypoxia is considered to drive the angiogenic response by upregulating angiogenic cytokines observed during wound healing. But this correlation has not been shown on a cell-to-cell basis in vivo because of limitations in measuring tissue PO2 at the cellular level. METHODS: Using punch biopsy wounds in rats as a wound healing model, the distributions of vascular endothelial growth factor, transforming growth factor-beta, tumor necrosis factor-alpha, and pimonidazole adducts (as a hypoxia marker) were followed immunohistochemically during the healing process. RESULTS: Hypoxia was absent on day 1 after wounding, even though angiogenesis and maximal expression of cytokines were observed in the wounds. Hypoxia peaked in the granulation tissue stage at day 4 and correlated with increased cellularity and cellular proliferation. Hypoxia started to decrease after day 4 and was limited to the remnant blood vessels and epithelial layer in the scar tissue. CONCLUSIONS: Induction of angiogenic cytokines early during wound healing may be due to triggering mechanisms other than hypoxia. Alternatively, the unique pattern of development and decline of cellular hypoxia as wound cellularity and proliferation regress suggest its involvement in initiating vascular regression during the later stages of healing.

Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis.

Tissue transglutaminase (TG) is an enzyme that stabilizes the structure of tissues by covalently ligating extracellular matrix molecules. Expression and localization of TG are not well established during wound healing. We performed punch biopsy wounds on anesthetized rats and monitored the wound healing process by histological and immunohistochemical methods. The TG antigen and activity are expressed at sites of neovascularization in the provisional fibrin matrix within 24 h of wounding. Endothelial cells, macrophages, and skeletal muscle cells expressed TG throughout the healing process. The TG antigen within the wound was active in vivo based on the detection of isopeptide bonds. The TG antigen increased four- to fivefold by day 3 postwounding and was proteolytically degraded. TG expression occurred in association with TGF-beta, TNF-alpha, IL-6, and VEGF production in the wound. Recombinant TG increased vessel length density (a measure of angiogenesis) when applied topically in rat dorsal skin flap window chambers. We have established that TG is an important tissue stabilizing enzyme that is active during wound healing and can function to promote angiogenesis.

Site-directed mutagenesis of the calcium-binding site of blood coagulation factor XIIIa.

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The K(act) values for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the K(act) values were not changed by mutagenesis. However, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu(485) and Glu(490) are Ca(2+)-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.

Tissue transglutaminase is expressed as a host response to tumor invasion and inhibits tumor growth.

A stable extracellular matrix (ECM) constitutes an important part of host response mechanism against tumor growth and invasion. Tissue transglutaminase (TG), a calcium-dependent enzyme, can cross-link all major ECM proteins to form a stable ECM, because these cross-links are resistant to proteolytic and mechanical damage. TG can also enhance stability and strength of the ECM by its ability to facilitate the activation of transforming growth factor-beta. We hypothesized that TG ECM-promoting abilities form an important part of the host response mechanism against tumor growth. Increased expression of TG was observed in the ECM of the host tumor interface of subcutaneously implanted rat mammary adenocarcinoma R3230 Ac. TG expression was also detected in the endothelial cells and macrophages. We also detected the cross-link product at the host tumor interface and within the tumor tissue, showing that TG was active. Western blots showed TG was degraded into three fragments of 55-, 50-, and 20-kDa forms. When recombinant wild-type TG was applied to R3230 Ac implanted in rat dorsal skin flap window chamber, it caused significant growth delay at day 7 compared with recombinant inactive TG controls. Collagen was detected in increased amounts in TG treated tumors, suggesting augmentation of production and stability of the ECM. We conclude that TG forms a distinct part of host response system against and acts to inhibit tumor growth.

Standardization of prothrombin fragment 1.2 measurement: effects of preanalytical variables and calibrator selection.

BACKGROUND: Immunoassays for prothrombin fragment 1.2 (F1.2) provide a specific measure of thrombin generation and offer potential value in detecting activation of the coagulation system and monitoring anticoagulant therapy. To standardize laboratory measurements of this analyte, it is important to define factors affecting interassay variability. OBJECTIVE: To determine the potential for standardization of F1.2 measurement by examining the effects of preanalytical variables and calibrator selection on F1.2 measurement. MATERIALS AND METHODS: Using three commercially available immunoassays, interassay and intra-assay correlations for F1.2 were determined using blood samples collected into heparin, citrate, and a solution of ethylenediaminetetraacetic acid, aprotinin, and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. In a cohort of patients, interassay correlations for F1.2 were determined using blood collected from an arterial catheter. Dose-response curves were generated for each manufacturer-supplied calibrator set by substitution into each of the previously untested competing immunoassays. RESULTS: F1.2 immunoassays with the same recommended specimen anticoagulant displayed stronger correlation than assays requiring different anticoagulants. Furthermore, a stronger interassay correlation was elicited by samples collected through an intra-arterial catheter as opposed to venipuncture. F1.2 calibrator sets differed quantitatively, with buffer-related matrix effects contributing to interassay variability. CONCLUSION: Analytical standardization of F1.2 immunoassays is possible when a common anticoagulant, blood collection method, and calibrator set are used.

Regulation of human tissue transglutaminase function by magnesium-nucleotide complexes. Identification of distinct binding sites for Mg-GTP and Mg-ATP.

Tissue transglutaminase (tTG) catalyzes a Ca(2+)-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 +/- 10 microM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 +/- 35 microM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 microM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 microM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.

The effects of epsilon-aminocaproic acid on fibrinolysis and thrombin generation during cardiac surgery.

UNLABELLED: Despite the efficacy of antifibrinolytic drugs in reducing bleeding after cardiac surgery, concerns remain regarding their potential to promote thrombosis. We examined the effect of the antifibrinolytic drug, epsilon-aminocaproic acid (EACA) on fibrinolysis and thrombin generation during cardiac surgery. Forty-one adults undergoing primary coronary artery bypass graft surgery requiring cardiopulmonary bypass (CPB) were prospectively randomized in a double-blind trial to receive either saline or EACA. A loading dose of 150 mg/kg EACA was given before anesthetic induction, followed by a 15 mg x kg(-1) x h(-1) infusion, which continued until 3 h after CPB. Plasma samples for the measurement of D-dimer, thrombin-antithrombin III, and soluble fibrin were obtained before surgery, 1 h on CPB, and 3 and 20 h after CPB. In the EACA group, fibrinolytic activity, as measured by D-dimer, was significantly decreased 3 h after CPB, (0.51 +/- 0.15 mg/L vs 1.13 +/- 0.14 mg/L, P < 0.005). Decreased fibrinolytic activity was accompanied by decreased bleeding in the EACA group (660 +/- 127 mL vs 931 +/- 113 mL, P < 0.05). No differences in the generation of thrombin or soluble fibrin were apparent between the two groups. Suppression of fibrinolytic activity in the absence of concomitant reductions in thrombin generation suggests that EACA could potentiate a hypercoagulable prethrombotic state in the perioperative setting. IMPLICATIONS: In a randomized, prospective trial of primary cardiac surgery, we demonstrated that the synthetic antifibrinolytic drug epsilon-aminocaproic acid suppresses fibrinolysis with no effects on thrombin generation. These results suggest the potential for synthetic antifibrinolytic drugs to induce a hypercoagulable prethrombotic state in the perioperative setting.

Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues.

Angiogenesis, the process of new vessels sprouting from the existing vasculature, is a critical process during early development. However, angiogenesis rarely occurs in the adult, except in response to cyclic hormonal stimulation in the ovary and uterus, in response to injury, and in response to pathological conditions such as tumorigenesis and diabetes mellitus. Tie2 (also known as Tek) is a novel endothelium-specific receptor tyrosine kinase, which has been demonstrated to be essential for the development of the embryonic vasculature; Tie2 knockout mice die by embryonic day 10.5 with specific defects in the formation of microvessels. Tie2 is downregulated later in embryogenesis, and its function in the adult has been relatively unexplored. To gain insight into the potential functions of Tie2 in the adult vasculature, Tie2 expression was examined in adult tissues undergoing angiogenesis and in quiescent tissues. Tie2 expression was localized by immunohistochemistry to the endothelium of neovessels in rat tissues undergoing angiogenesis during hormonally stimulated follicular maturation and uterine development and in healing skin wounds. Immunoprecipitation and RNase protection assay demonstrated upregulation of Tie2 protein and mRNA in rat and mouse skin wounds, respectively. Moreover, Tie2 immunoprecipitated from skin wounds was tyrosine-phosphorylated, indicating active downstream signaling. Surprisingly, Tie2 was also expressed in the entire spectrum of the quiescent vasculature (arteries, veins, and capillaries) in a wide range of adult tissues, and Tie2 immunoprecipitated from quiescent adult tissues was also tyrosine-phosphorylated. Together, these results suggest a dual function for Tie2 in adult tissues involving both angiogenesis and vascular maintenance.

Analysis of factor XIII substrate specificity using recombinant human factor XIII and tissue transglutaminase chimeras.

Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins. FXIII requires thrombin for activation and cross-links the gamma chains of fibrin(ogen) more efficiently than the Aalpha chains. On the other hand, tTG is thrombin-independent and forms predominantly Aalpha and Aalpha-gamma chain complexes. Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (Hettasch, J. M., and Greenberg, C. S. (1994) J. Biol. Chem. 269, 28309-28313). To determine to what extent the primary amino acid sequence within exon 7 defines substrate specificity, exon 7 of FXIII was replaced with the corresponding exon of tTG using gene splicing by overlap extension. Other work from this laboratory (Achyuthan, K. E., Slaughter, T. F., Santiago, M. A., Enghild, J. J., and Greenberg, C. S. (1993) J. Biol. Chem. 268, 21284-21292) using synthetic peptides identified two other domains that might play a role in substrate recognition (located in exons 3 and 5). Therefore, recombinant chimeras of FXIII/tTG were also created in which these two exons were exchanged. FXIII, tTG, and chimeras 3, 5, and 7 were expressed in Escherichia coli, purified, and the nature of the fibrin cross-linking pattern of these five proteins was determined by immunoblot analysis. FXIII preferentially formed the gamma-gamma dimer, whereas tTG formed Aalpha-gamma complexes. Chimera 7 formed Aalpha-gamma complexes that resembled the cross-linking pattern of tTG. This finding demonstrates that the primary amino acid sequence of exon 7 of tTG confers some of the specificity for the Aalpha and Aalpha-gamma cross-link pattern characteristic of tTG. Chimera 5 exhibited reduced cross-linking activity (50% of FXIII activity) but still retained preference for formation of the gamma-gamma dimer, whereas chimera 3 was not active. In conclusion, exchanging the primary amino acid sequence of the active site exon of human FXIII with that of human tTG modifies the enzyme such that the fibrin cross-linking pattern more closely resembles that of tTG (Aalpha and Aalpha-gamma complexes) instead of FXIII (gamma-gamma dimers).