Using cadaveric simulation to introduce the concept and skills required to start performing transanal total mesorectal excision.

AIM: The aim was to document the outcomes of surgeons attending a cadaveric simulation course designed to provide an introduction to transanal total mesorectal excision (TaTME). METHOD: This was a prospective observational study documenting the outcomes from classroom and wet lab activities. Follow-up questionnaires were used to monitor clinical activity after the course. RESULTS: Outcomes of 65 delegates from 12 different countries attending seven cadaveric simulation courses are described. Median time to insert and close the rectal purse-string was 15 min (range 7-50 min) and median time to complete the transanal mesorectal dissection was 105 min (range 60-260 min). Objective assessment of specimen quality showed that 42% of specimens were complete, 47% nearly complete and 11% were incomplete. Failure of the intraluminal rectal purse-string was the most common difficulty encountered. Within 6 months of attending the course, nearly half (26/55; 47%) of the surgeons who responded had performed between 1 and 13 TaTMEs. Only 8/26 (31%) of the surgeons had arranged mentoring for their first case. CONCLUSION: This training model provides high levels of trainee satisfaction and the knowledge and technical skills to enable them to start performing TaTME. There is still work to do to provide adequate supervision and mentorship for surgeons early on their learning curve that is essential for the safe introduction of this new technique.

Underexpression of tumour suppressor LKB1 in clear cell renal cell carcinoma is common and confers growth advantage in vitro and in vivo.

BACKGROUND: Evidence suggests that dysregulation of energy-sensing pathways closely associates with renal cell carcinoma (RCC) development. The metabolic regulation is largely controlled by 5'-AMP activated protein kinase (AMPK) which is activated through phosphorylation by LKB1. METHODS: The expression of LKB1 was determined by reverse transcription-PCR using 10 clinical clear cell RCC (ccRCC) samples and their adjacent normal renal parenchyma, and by immunohistochemical staining of two tissue microarrays containing 201 ccRCC and 26 normal kidney samples. Expression of LKB1 was knocked down in human ccRCC 786-O cells (shLKB1) and compared with cells expressing scrambled control shRNA (shControl). AMPK signalling, proliferation, invasion, and VEGF secretion was measured. The cells were subcutaneously injected into mice to determine tumour growth in vivo. RESULTS: At the protein and transcript levels, a significant reduction in LKB1 expression in tumour compared with normal tissue was found. In vitro, knockdown of LKB1 resulted in reduced AMPK signalling and increased cellular proliferation, invasion, and VEGF secretion compared with shControl cells. In vivo, growth of shLKB1 ccRCC xenografts in nude mice was significantly increased compared with shControl xenografts. CONCLUSION: Collectively, our results suggest that LKB1 acts as a tumour suppressor in most sporadic cases of ccRCC and that underexpression of LKB1 is a common event in the disease.

Prenatal stress enhances severity of atherosclerosis in the adult apolipoprotein E-deficient mouse offspring via inflammatory pathways.

Atherosclerosis is the underlying cause of cardiovascular disease and stroke. Endothelial cell dysfunctions are early events in atherosclerosis, resulting in the recruitment of circulating monocytes. The immune system can elicit an inflammatory response toward the atherosclerotic lesion, thereby accelerating lesion growth. Risk factors for atherosclerosis include hypertension, smoking, stress perception or low birth weight. As prenatal stress challenge decreases the birth weight and affects the offspring's postnatal immune response, we aimed to investigate whether prenatal stress contributes to the development of atherosclerosis in mice. Syngenic pregnant apolipoprotein E-deficient (apoE-/-) dams were exposed to sound stress on gestation days 12.5 and 14.5. The presence and size of atherosclerotic plaques in the offspring at the age of 15 weeks was evaluated by histomorphology, accompanied by flow cytometric analysis of the frequency and phenotype of monocytes/macrophages and regulatory T (Treg) cells in the blood. Further, cytokine secretion of peripheral blood lymphocytes was analyzed. In response to prenatal stress challenge, an increased frequency of large atherosclerotic plaques was detectable in apoE-/- offspring, which was particularly profound in females. Prenatal stress also resulted in alterations of the offspring's immune response, such as a decreased frequency of Treg cells in blood, alterations of macrophage populations in blood and an increased secretion of inflammatory cytokines. We provide novel evidence that prenatally stressed adult offspring show an increased severity of atherosclerosis. As Treg cells are key players in dampening inflammation, the observed increase in atherosclerosis may be due to the lack of Treg cell frequency. Future interdisciplinary research is urgently required to understand the developmental origin of prenatal stress-induced atherosclerosis. The availability of our model may facilitate and foster such research endeavors.

Laparoscopic total mesorectal excision following long course chemoradiotherapy for locally advanced rectal cancer.

PURPOSE: Laparoscopic total mesorectal excision (TME) of locally advanced rectal cancer after long-course chemoradiotherapy (LCRT) is surgically and oncologically challenging. We have assessed the feasibility, timing, and short-term oncological outcome of laparoscopic TME after LCRT. METHODS: Between 2004 and 2006, 30 patients were selected for LCRT based on clinical examination and MRI. Patients received 3/4 field radiotherapy, 45-50.4 Gy in 25-28 fractions during 5 weeks with either 5-fluorouracil or Uftoral. Clinical assessments were made 4 weeks after completion of radiotherapy and then 2 weekly with sequential 4 weekly MRI, to individualize the timing of surgery at maximal response. Laparoscopic TME was performed using a standard technique. RESULTS: Thirty patients received LCRT and 26 patients (21 men; median age, 63 years) underwent laparoscopic TME at 11 weeks (median) after LCRT. Median operating time was 270 min. Sixteen patients had LAR and ten had APR. There were three conversions. Three patients developed anastomotic leak (18.7%): one was managed conservatively and one patient died of septicemia. Morbidity was seen in 19% of patients. There were 25 (96%) R0 resections with a complete response in 5 (19%) cases and microscopic tumor in lakes of mucin (Tmic) in another 6 (23%). Two patients (7.6%) developed local recurrence (median follow up, 34 months). The median time interval between radiotherapy and surgery was 11 (range, 7-13) weeks, which was based on serial MRI scans after LCRT. CONCLUSIONS: Laparoscopic TME after LCRT is feasible and safe both oncologically and surgically. Serial MRI helps to determine the optimum timing of surgery.

Activated protein C modulates inflammation, apoptosis and tissue factor procoagulant activity by regulating endoplasmic reticulum calcium depletion in blood monocytes.

BACKGROUND: The endoplasmic reticulum (ER) is responsible for the synthesis and folding of secretory, transmembrane and ER-resident proteins. Conditions that impair protein folding or overwhelm its protein folding capacity disrupt ER homeostasis, thereby causing ER stress. ER stress-induced apoptosis and inflammation are involved in the pathogenesis of inflammatory diseases. Activated protein C (APC) inhibits inflammation and apoptosis in monocytes, and this may partly explain the protective effects of APC treatment in severe sepsis. However, the precise molecular pathways by which APC modulates these effects remain unknown. OBJECTIVES: To investigate whether APC modulates the ER stress response in human monocytes. METHODS: We treated monocytes with ER stress-inducing agents in the presence or absence of APC to determine the effect on this response. Protein and mRNA levels were determined by immunoblotting and real-time PCR, respectively. Enzyme assays and flow cytometry were used to determine the role of APC in this model. RESULTS: In thapsigargin (Tg)-treated cells, APC dampened unfolded protein response activation, as indicated by reduced levels of the 78-kDa glucose-regulated protein (GRP78), in an endothelial protein C receptor-independent and protease-activated receptor-1-independent manner. Consistent with this, APC decreased phosphorylated eukaryotic translational initiation factor 2α and C/EBP homologous protein levels induced by Tg. APC inhibited Tg-induced ER Ca(2+) flux and reactive oxygen species generation. Functionally, APC diminished Tg-induced caspase-3 activity and degradation of the nuclear factor kappaB inhibitor IκBα. Furthermore, APC dampened the induction of tissue factor procoagulant activity facilitated by Tg. CONCLUSIONS: These studies suggest that APC modulates the adverse effects of ER Ca(2+) depletion in human monocytes.

A novel use of T-tag sutures for the safe creation and closure of the NOTES gastrotomy using a hybrid technique.

Transgastric Natural Orifice Translumenal Endoscopic Surgery (NOTES) procedures are presently limited by the lack of a reliable method for creating and closing the gastrotomy created during the procedure. Furthermore, due to difficulties with the NOTES technique, the majority of NOTES procedures are presently performed in a "hybrid" fashion in which some degree of laparoscopic assistance is used alongside the NOTES approach. We describe a hybrid approach with a minimal laparoscopic component allowing a very controlled gastrotomy creation and closure. This technique would also allow laparoscopic suturing through a single, small cannula.

Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease, including ischemic heart disease, stroke, and peripheral vascular disease. Mutations in the enzymes responsible for homocysteine metabolism, particularly cystathionine beta-synthase (CBS) or 5,10-methylenetetrahydrofolate reductase (MTHFR), result in severe forms of HHcy. Additionally, nutritional deficiencies in B vitamin cofactors required for homocysteine metabolism, including folic acid, vitamin B6 (pyridoxal phosphate), and/or B12 (methylcobalamin), can induce HHcy. Studies using animal models of genetic- and diet-induced HHcy have recently demonstrated a causal relationship between HHcy, endothelial dysfunction, and accelerated atherosclerosis. Dietary enrichment in B vitamins attenuates these adverse effects of HHcy. Although oxidative stress and activation of proinflammatory factors have been proposed to explain the atherogenic effects of HHcy, recent in vitro and in vivo studies demonstrate that HHcy induces endoplasmic reticulum (ER) stress, leading to activation of the unfolded protein response (UPR). This review summarizes the current role of HHcy in endothelial dysfunction and explores the cellular mechanisms, including ER stress, that contribute to atherothrombosis.

Dietary supplementation with methionine and homocysteine promotes early atherosclerosis but not plaque rupture in ApoE-deficient mice.

Hyperhomocysteinemia is an independent risk factor for atherothrombosis. However, causality is unproven, and it remains unknown whether hyperhomocysteinemia promotes atherosclerosis, plaque rupture, and/or thrombosis. We evaluated the short- and long-term effects of hyperhomocysteinemia on plaque size and structure in 99 atherosclerosis-prone apolipoprotein E-deficient mice. Hyperhomocysteinemia was induced by methionine (Met) or homocysteine (HcyH) supplementation: low Met (+11 g Met/kg food), high Met (+33 g Met/kg food), low HcyH (0.9 g HcyH/L drinking water), and high HcyH (1.8 g HcyH/L drinking water). Met and HcyH supplementation significantly raised plasma total homocysteine levels by 4- to 16-fold above those observed in mice fed a control diet (up to 146.1 micromol/L). Compared with controls, aortic root plaque size was significantly larger in supplemented groups after 3 months (56% and 173% larger in high-Met and high-HcyH, respectively) but not after 12 months. Hyperhomocysteinemia was associated with an increase in the amount of collagen in plaques after both 3 and 12 months. Mechanical testing of the tail tendons revealed no weakening of collagen after 12 months of hyperhomocysteinemia. Many plaques in both control and supplemented mice appeared rupture prone morphologically, but all aortic root plaques and all but 1 coronary plaque had an intact surface without rupture or thrombosis. Thus, diet-induced hyperhomocysteinemia promotes early atherosclerosis and plaque fibrosis but does not, even in the long term, weaken collagen or induce plaque rupture.

Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways.

Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine beta-synthase. However, the mechanism by which homocysteine promotes the development and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regulatory element-binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intracellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by overexpression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the activation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secretion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia.

Placental transforming growth factor-beta is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression.

The p53 tumor suppressor gene and members of the transforming growth factor-beta (TGF-beta) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-beta superfamily, designated placental TGF-beta (PTGF-beta), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-beta is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-beta promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-beta promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-beta overexpression from a recombinant adenoviral vector (AdPTGF-beta) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50-60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-beta overexpression was seen to induce both G(1) cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-beta superfamily and implicate PTGF-beta as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.

Expression and synthesis of alternatively spliced variants of Dp71 in adult human brain.

Transcripts encoding the 70-75 kDa C-terminal protein product of the dystrophin gene (Dp71) are alternatively spliced to generate multiple protein products in a number of adult human tissues. In this report, reverse transcriptase-polymerase chain reaction was used to clone and characterize a subpopulation of truncated Dp71 transcripts in adult human brain tissue which did not contain exons 71-74, resulting in an in-frame deletion of 330 bp encoding the syntrophin-binding domain. These truncated Dp71 transcripts are also alternatively spliced for exon 78. Immunoblot analysis, using dystrophin-specific C-terminal antibodies directed against epitopes in either exon 77 (MANDRA1), or 78 (1461), identified full-length dystrophin, Dp140 and Dp71, in total protein lysates from adult human brain tissue. In addition, a minor immunoreactive protein of approximately 58 kDa was also identified (designated Dp71 big up tri, open(110)). The observation that a monoclonal antibody directed against epitopes within exons 73-74 (MANEX7374A) failed to detect this 58 kDa protein provides definitive evidence that Dp71 big up tri, open(110) is derived from Dp71 transcripts deleted for the syntrophin-binding domain. These results, as well as previous findings, demonstrate that alternative splicing of Dp71 in the human brain generates a variety of mRNA transcripts encoding distinct protein variants of Dp71, and further supports the use of exon-specific antibodies in characterizing these variants. The presence of these Dp71 protein variants in brain tissue points to their interaction with various cellular proteins and their involvement in different cellular functions.

Radioimmunodetection: technical problems and methods of improvement.

Radioimmunodetection (RAID) is a technique which uses radiolabelled antibodies to visualize tumours, taking advantage of antigens preferentially expressed by malignant tissue. Gamma radiation emitted by radioisotopes can be detected using an external gamma camera (RAID), or intraoperatively with a hand-held Geiger counter (radioimmunoguided surgery, RIGS). RAID has significant inherent problems. Many have been overcome as a result of nearly 50 years of research, and others still remain as obstacles precluding the routine use of the technique. This article summarizes the technical limitations of RAID and outlines the relative successes of the methods evolved to overcome them.

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Arg(193), demonstrated a approximately 130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha-thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation approximately 100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.

Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells.

Alterations in the cellular redox potential by homocysteine promote endothelial cell (EC) dysfunction, an early event in the progression of atherothrombotic disease. In this study, we demonstrate that homocysteine causes endoplasmic reticulum (ER) stress and growth arrest in human umbilical vein endothelial cells (HUVEC). To determine if these effects reflect specific changes in gene expression, cDNA microarrays were screened using radiolabeled cDNA probes generated from mRNA derived from HUVEC, cultured in the absence or presence of homocysteine. Good correlation was observed between expression profiles determined by this method and by Northern blotting. Consistent with its adverse effects on the ER, homocysteine alters the expression of genes sensitive to ER stress (ie, GADD45, GADD153, ATF-4, YY1). Several other genes observed to be differentially expressed by homocysteine are known to mediate cell growth and differentiation (ie, GADD45, GADD153, Id-1, cyclin D1, FRA-2), a finding that supports the observation that homocysteine causes a dose-dependent decrease in DNA synthesis in HUVEC. Additional gene profiles also show that homocysteine decreases cellular antioxidant potential (glutathione peroxidase, NKEF-B PAG, superoxide dismutase, clusterin), which could potentially enhance the cytotoxic effects of agents or conditions known to cause oxidative damage. These results successfully demonstrate the use of cDNA microarrays in identifying homocysteine-respondent genes and indicate that homocysteine-induced ER stress and growth arrest reflect specific changes in gene expression in human vascular EC.

Dystrophin isoforms DP71 and DP427 have distinct roles in myogenic cells.

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two dystrophin isoforms are expressed, Dp427 and Dp71. To characterize the function of Dp71 in myogenesis, we have examined the expression of Dp71 in myogenic cells. The localization of Dp71 in these cells is distinct from the localization of Dp427. Whereas Dp427 localizes to focal adhesions and surface membrane during myogenesis, Dp71 localizes to stress fiberlike structures in myogenic cells. Biochemical fractionation of myogenic cells demonstrates that Dp71 cosediments with the actin bundles thus confirming this interaction. Furthermore, transfection of C2C12 myoblasts with constructs encoding Dp71 fused to green fluorescent protein targeted the protein to the actin microfilament bundles. These results demonstrate involvement of Dp71 with the actin cytoskeleton during myogenesis and suggest a role for Dp71 that is distinct from Dp427.

Homocysteine-dependent alterations in mitochondrial gene expression, function and structure. Homocysteine and H2O2 act synergistically to enhance mitochondrial damage.

Mitochondrial abnormalities have been identified in hepatocytes of patients with hyperhomocysteinemia and in endothelial cells from the aortas of rats with diet-induced hyperhomocysteinemia. However, the mechanism by which homocysteine affects mitochondria is unknown. In this report, homocysteine-induced expression of the mitochondrial electron transport chain gene, cytochrome c oxidase III/ATPase 6,8 (CO3/ATPase 6,8), was identified in a human megakaryocytic cell line DAMI using mRNA differential display. Steady-state mRNA levels of CO3/ATPase 6,8, as well as other mitochondrial transcripts, were increased in DAMI cells by homocysteine in a concentration- and time-dependent manner. Despite an increase in mitochondrial RNA levels and changes in mitochondrial ultrastructure, no effect on either cell growth or mitochondrial respiration rates was observed in DAMI cells exposed to homocysteine at concentrations up to 1 mM. In contrast, 1 mM homocysteine in the presence of Cu2+, which is known to generate H2O2, significantly decreased mitochondrial RNA levels, caused gross morphological changes in mitochondrial ultrastructure, and inhibited both cell growth and mitochondrial respiration rates. However, precursors of cellular glutathione and preexposure to heat shock blocked the decrease in mitochondrial RNA levels caused by homocysteine and Cu2+. The observations that (i) homocysteine and H2O2, but not H2O2 alone, caused a decrease in mitochondrial RNA levels, (ii) intracellular levels of H2O2 were significantly increased in the presence of homocysteine and Cu2+, and (iii) catalase, but not free radical scavengers, prevented a decrease in mitochondrial RNA levels, provide evidence that homocysteine and H2O2 act synergistically to cause mitochondrial damage. Furthermore, our findings suggest that intracellular glutathione and heat shock proteins play a role in protecting mitochondria against the adverse effects elicited by homocysteine and H2O2.

Characterization of the stress-inducing effects of homocysteine.

The mechanism by which homocysteine causes endothelial cell (EC) injury and/or dysfunction is not fully understood. To examine the stress-inducing effects of homocysteine on ECs, mRNA differential display and cDNA microarrays were used to evaluate changes in gene expression in cultured human umbilical-vein endothelial cells (HUVEC) exposed to homocysteine. Here we show that homocysteine increases the expression of GRP78 and GADD153, stress-response genes induced by agents or conditions that adversely affect the function of the endoplasmic reticulum (ER). Induction of GRP78 was specific for homocysteine because other thiol-containing amino acids, heat shock or H2O2 did not appreciably increase GRP78 mRNA levels. Homocysteine failed to elicit an oxidative stress response in HUVEC because it had no effect on the expression of heat shock proteins (HSPs) including HSP70, nor did it activate heat shock transcription factor 1. Furthermore homocysteine blocked the H2O2-induced expression of HSP70. In support of our findings in vitro, steady-state mRNA levels of GRP78, but not HSP70, were elevated in the livers of cystathionine beta-synthase-deficient mice with hyperhomocysteinaemia. These studies indicate that the activation of stress response genes by homocysteine involves reductive stress leading to altered ER function and is in contrast with that of most other EC perturbants. The observation that homocysteine also decreases the expression of the antioxidant enzymes glutathione peroxidase and natural killer-enhancing factor B suggests that homocysteine could potentially enhance the cytotoxic effect of agents or conditions known to cause oxidative stress.

Localization of the thrombin-binding domain on prothrombin fragment 2.

Co-crystallographic studies have shown that the interaction of human prothrombin fragment 2 (F2) with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin-binding domain on F2. F2, rF2-(1-116), rF2-(55-116), and sF2-(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic COOH-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2-(63-90), or the COOH-terminal connecting peptide, sF2-(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the COOH-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2-(63-116) bind saturably to fluorescently labeled active site-blocked thrombin with Kd values of 4.1 and 51.3 microM, respectively. The affinity of sF2-(63-116) for thrombin increases about 5-fold (Kd = 10 microM) when Val at position 78 is substituted with Glu. F2 and sF2-(63-116) bind to exosite II on thrombin because both reduce the heparin-catalyzed rate of thrombin inhibition by antithrombin approximately 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2-(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently labeled hirudin54-65 from active site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion, reflecting their effects on the active site and/or exosite I. These studies provide further insight into the regions of F2 that evoke functional changes in thrombin.