Novel NMP split liver model recapitulates human IRI and demonstrates ferroptosis modulators as a new therapeutic strategy.

BACKGROUND: Almost 9%of deceased donor livers are discarded as marginal donor livers (MDL) due to concern of severe ischemia reperfusion injury (IRI). Emerging data supports ferroptosis (iron regulated hepatocellular death) as an IRI driver, however lack of robust preclinical model limits therapeutic testing. In this manuscript we describe the development of a novel rigorous internal control system utilizing normothermic perfusion of split livers to test ferroptosis regulators modulating IRI. METHODS: Upon institutional approval, split human MDLs were placed on our normothermic perfusion machine, Perfusion Regulated Organ Therapeutics with Enhanced Controlled Testing (PROTECT), pumping arterial and portal blood. Experiment 1 compared right (UR) and left (UL) lobes to validate PROTECT. Experiment 2 assessed ferroptosis regulator Deferoxamine in Deferoxamine Agent Treated (DMAT) vs. No Agent Internal Control (NAIC) lobes. Liver serology, histology, and ferroptosis genes were assessed. RESULTS: Successful MDL perfusion validated PROTECT with no ALT or AST difference between UR and UL (∆ALT UR: 235, ∆ALT UL: 212; ∆AST UR: 576, ∆AST UL: 389). Liver injury markers increased in NAIC vs. DMAT (∆ALT NAIC: 586, ∆ALT DMAT: -405; ∆AST NAIC: 617, ∆AST DMAT: -380). UR and UL had similar expression of ferroptosis regulators RPL8,HO-1 and HIFα. Significantly decreased intrahepatic iron (p = .038), HO-1 and HIFα in DMAT (HO-1 NAIC: 6.93, HO-1 DMAT: 2.74; HIFαNAIC: 8.67, HIFαDMAT: 2.60)and no hepatocellular necrosis or immunohistochemical staining (Ki67/Cytokeratin-7) differences were noted. CONCLUSION: PROTECT demonstrates the therapeutic utility of a novel normothermic perfusion split liver system for drug discovery and rapid translatability of therapeutics, driving a paradigm change in organ recovery and transplant medicine. Our study using human livers, provides preliminary proof of concept for the novel role of ferroptosis regulators in driving IRI.

Developing a Novel Ambulatory Total Parenteral Nutrition-Dependent Short Bowel Syndrome Animal Model.

BACKGROUND: Short bowel syndrome (SBS) results from extensive bowel resection. Patients with SBS require total parenteral nutrition (TPN) for survival. Understanding mechanisms contributing to TPN-associated liver injury and gut atrophy are critical in developing SBS therapies. Existing SBS models using tethered animals have significant limitations and are unlike ambulatory human SBS patients. We hypothesized that we could induce SBS in piglets and develop an ambulatory TPN-SBS model. MATERIAL AND METHODS: Eighteen neonatal pigs received duodenal and jugular catheters. They were fitted with a jacket holding TPN and a miniaturized pump. Six piglets had 90% small bowel resection and catheter placement (SBS group). Non-SBS piglets were randomized into enteral nutrition (EN) or TPN. RESULTS: Bowel resection was successfully accomplished in SBS animals. Weight gain was similar in all groups. SBS animals had increased serum bilirubin compared to EN. Mean conjugated bilirubin ± SD was 0.045 ± 0.01 for EN, (P = 0.03 EN versus TPN and P = 0.03 SBS versus EN) and 1.09 ± 1.25 for TPN, (P = 0.62 TPN versus SBS). Gut density was reduced in the TPN group compared to EN and SBS groups. Mean gut density ± SD was 0.11 ± 0.04 for TPN (P = 0.0004 TPN versus SBS and P = 0.00007 TPN versus EN) and not statistically different for EN versus SBS (P = 0.32). CONCLUSIONS: We created a novel, ambulatory TPN-SBS model using piglets, mimicking long-term TPN delivery in human SBS patients. Our model demonstrated TPN-related conjugated hyperbilirubinemia and compensatory gut hypertrophy, as noted in humans with SBS. This model holds great potential for future research.

Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor.

The extracellular nuclear proteins, histone H4 (H4) and high mobility group box 1 (HMGB1), released by injured cells during the activation of inflammation and coagulation pathways provoke potent inflammatory responses through interaction with pathogen-related pattern recognition receptors (ie, Toll-like receptors [TLRs] and receptor for advanced glycation end products [RAGE]) present on vascular and innate immune cells. Inorganic polyphosphate (polyP) has emerged as a key modulator of coagulation and inflammation. Here, we demonstrate that polyP binds to both H4 and HMGB1 with high affinity, thereby dramatically potentiating their proinflammatory properties in cellular and in vivo models. By using small interfering RNA knockdowns, pharmacologic inhibitors and extracellular domains of the receptors TLR2, TLR4, RAGE, and P2Y1 as competitive inhibitors, we demonstrate that polyP amplifies H4- and HMGB1-mediated inflammatory signaling in human umbilical vein endothelial cells specifically through interaction with the RAGE and P2Y1 receptors, thereby eliciting intracellular Ca(2+) release. Finally, we demonstrate that the natural anticoagulant protease, activated protein C, potently inhibits polyP-mediated proinflammatory effects of both nuclear proteins in cellular and in vivo systems.

Expression and Characterization of Gly-317 Variants of Factor IX Causing Variable Bleeding in Hemophilia B Patients.

We recently identified two hemophilia B patients who carried Gly-317 to Arg (FIX-G317R) or Gly-317 to Glu (FIX-G317E) substitutions in their FIX gene. The former mutation caused severe and the latter moderate bleeding in afflicted patients. To understand the molecular basis for the variable clinical manifestation of Gly-317 mutations, we prepared recombinant G317R and G317E derivatives of FIX and compared their kinetic properties to those of recombinant wild-type FIX in appropriate assay systems. Both physiological activators, factor XIa and extrinsic Tenase (factor VIIa-tissue factor), activated both zymogen variants with an ∼1.5-fold elevated K(m); however, extrinsic Tenase activated FIX-G317E with an ∼2-fold improved k(cat). By contrast to zymogen activation, the catalytic activities of both FIXa-G317R and FIXa-G317E enzymes toward the natural substrate, factor X, were dramatically (>4 orders of magnitude) impaired, but their apparent affinity for interaction with factor VIIIa was only slightly (<2-fold) decreased. Further studies revealed that the reactivity of FIXa-G317R and FIXa-G317E with antithrombin has been impaired 10- and 13-fold, respectively, in the absence and 166- and 500-fold, respectively, in the presence of pentasaccharide. As expected, the clotting activities of FIX variants could not be measured by the aPTT assay. These results implicate a critical role for Gly-317 in maintaining normal catalytic function for FIX/FIXa in the clotting cascade. The results further suggest that improved k(cat) of FIX-G317E activation in the extrinsic pathway together with dramatically impaired reactivity of FIXa-G317E with antithrombin may account for the less severe bleeding phenotype of a hemophilia B patient carrying the FIX-G317E mutation.

Characterization of the protein Z-dependent protease inhibitor interactive-sites of protein Z.

BACKGROUND: Protein Z (PZ) has been reported to promote the inactivation of factor Xa (FXa) by PZ-dependent protease inhibitor (ZPI) by about three orders of magnitude. Previously, we prepared a chimeric PZ in which its C-terminal pseudo-catalytic domain was grafted on FX light-chain (Gla and EGF-like domains) (PZ/FX-LC). Characterization of PZ/FX-LC revealed that the ZPI interactive-site is primarily located within PZ pseudo-catalytic domain. Nevertheless, the cofactor function and apparent Kd of PZ/FX-LC for interaction with ZPI remained impaired ~6-7-fold, suggesting that PZ contains a ZPI interactive-site outside pseudo-catalytic domain. X-ray structural data indicates that Tyr-240 of ZPI interacts with EGF2-domain of PZ. Structural data further suggests that 3 other ZPI surface loops make salt-bridge interactions with PZ pseudo-catalytic domain. To identify ZPI interactive-sites on PZ, we grafted the N-terminal EGF2 subdomain of PZ onto PZ/FX-LC chimera (PZ-EGF2/FX-LC) and also generated two compensatory charge reversal mutants of PZ pseudo-catalytic domain (Glu-244 and Arg-212) and ZPI surface loops (Lys-239 and Asp-293). METHODS: PZ chimeras were expressed in mammalian cells and ZPI derivatives were expressed in Escherichia coli. RESULTS: The PZ EGF2 subdomain fusion restored the defective cofactor function of PZ/FX-LC. The activities of PZ and ZPI mutants were all impaired if assayed individually, but partially restored if the compensatory charge reversal mutants were used in the assay. CONCLUSIONS: PZ EGF2 subdomain constitutes an interactive-site for ZPI. Data with compensatory charge reversal mutants validates structural data that the identified residues are part of interactive-sites. GENERAL SIGNIFICANCE: Insight is provided into mechanisms through which specificity of ZPI-PZ-FXa complex formation is determined.

Variable contributions of basic residues forming an APC exosite in the binding and inactivation of factor VIIIa.

Basic residues contained in the 39-, 60-, and 70-80-loops of activated protein C (APC) comprise an exosite that contributes to the binding and subsequent proteolytic inactivation of factor (F) VIIIa. Surface plasmon resonance (SPR) showed that WT APC bound to FVIII light chain (LC) and the FVIIIa A1/A3C1C2 dimer with equivalent affinity (Kd = 525 and 546 nM, respectively). These affinity values may reflect binding interactions to the acidic residue-rich a1 and a3 segments adjacent to A1 domain in the A1/A3C1C2 and A3 domain in LC, respectively. Results from SPR, using a panel of APC exosite variants where basic residues were mutated, in binding to immobilized FVIIIa A1/A3C1C2 or LC indicated ~4-10-fold increases in the Kd values relative to WT for several of the variants including Lys39Ala, Lys37-Lys38-Lys39/Pro-Gln-Glu, and Arg67Ala. On the other hand, a number of APC variants including Lys38Ala, Lys62Ala, and Lys78Ala showed little if any change in binding affinity to the FVIII substrates. FXa generation assays and Western blotting, used to monitor rates of FVIIIa inactivation and proteolysis at the primary cleavage site in the cofactor (Arg(336)), respectively, showed marked rate reductions relative to WT for the Lys39Ala, Lys37-Lys38-Lys39/Pro-Gln-Glu, Arg67Ala, and Arg74Ala variants. Furthermore, kinetic analysis monitoring FVIIIa inactivation by APC variants at varying FVIIIa substrate concentration showed ~2.6-4.4-fold increases in Km values relative to WT. These results show a variable contribution of basic residues comprising the APC exosite, with significant contributions from Lys39, Arg67, and Arg74 to forming a FVIIIa-interactive site.

Role of Effective Policy and Screening in Managing Pediatric Nutritional Insecurity as the Most Important Social Determinant of Health Influencing Health Outcomes.

Social Determinants of Health (SDOH) impact nearly half of health outcomes, surpassing the influence of human behavior, clinical care, and the physical environment. SDOH has five domains: Economic Stability, Education Access and Quality, Health Care Access and Quality, Neighborhood and Built Environment, and Social and Community Context. Any adversity arising out of these interlinked domains predominantly affects children due to their greater susceptibility, and the adverse outcomes may span generations. Unfavorable SDOH may cause food insecurity, malnutrition, unbalanced gut microbiome, acute and chronic illnesses, inadequate education, unemployment, and lower life expectancy. Systematic screening by health care workers and physicians utilizing currently available tools and questionnaires can identify children susceptible to adverse childhood experiences, but there is a deficiency with respect to streamlined approach and institutional support. Additionally, current ameliorating supplemental food programs fall short of pediatric nutritional requirements. We propose a nutrition-based Surveillance, Screening, Referral, and Reevaluation (SSRR) plan encompassing a holistic approach to SDOH with a core emphasis on food insecurity, coupled with standardizing outcome-based interventions. We also propose more inclusive use of Food Prescription Programs, tailored to individual children's needs, with emphasis on education and access to healthy food.

Gut Microbiota Modulation of Short Bowel Syndrome and the Gut-Brain Axis.

Short bowel syndrome (SBS) is a condition that results from a reduction in the length of the intestine or its functional capacity. SBS patients can have significant side effects and complications, the etiology of which remains ill-defined. Thus, facilitating intestinal adaptation in SBS remains a major research focus. Emerging data supports the role of the gut microbiome in modulating disease progression. There has been ongoing debate on defining a "healthy" gut microbiome, which has led to many studies analyzing the bacterial composition and shifts that occur in gastrointestinal disease states such as SBS and the resulting systemic effects. In SBS, it has also been found that microbial shifts are highly variable and dependent on many factors, including the anatomical location of bowel resection, length, and structure of the remnant bowel, as well as associated small intestinal bacterial overgrowth (SIBO). Recent data also notes a bidirectional communication that occurs between enteric and central nervous systems called the gut-brain axis (GBA), which is regulated by the gut microbes. Ultimately, the role of the microbiome in disease states such as SBS have many clinical implications and warrant further investigation. The focus of this review is to characterize the role of the gut microbiota in short bowel syndrome and its impact on the GBA, as well as the therapeutic potential of altering the microbiome.

Identification of exosite residues of factor Xa involved in recognition of PAR-2 on endothelial cells.

Recent results have indicated that factor Xa (FXa) cleaves protease-activated receptor 2 (PAR-2) to elicit protective intracellular signaling responses in endothelial cells. In this study, we investigated the molecular determinants of the specificity of FXa interaction with PAR-2 by monitoring the cleavage of PAR-2 by FXa in endothelial cells transiently transfected with a PAR-2 cleavage reporter construct in which the extracellular domain of the receptor was fused to cDNA encoding for alkaline phosphatase. Comparison of the cleavage efficiency of PAR-2 by a series of FXa mutants containing mutations in different surface loops indicated that the acidic residues of 39-loop (Glu-36, Glu-37, and Glu-39) and the basic residues of 60-loop (Lys-62 and Arg-63), 148-loop (Arg-143, Arg-150, and Arg-154), and 162-helix (Arg-165 and Lys-169) contribute to the specificity of receptor recognition by FXa on endothelial cells. This was evidenced by significantly reduced activity of mutants toward PAR-2 expressed on transfected cells. The extent of loss in the PAR-2 cleavage activity of FXa mutants correlated with the extent of loss in their PAR-2-dependent intracellular signaling activity. Further characterization of FXa mutants indicated that, with the exception of basic residues of 162-helix, which play a role in the recognition specificity of the prothrombinase complex, none of the surface loop residues under study makes a significant contribution to the activity of FXa in the prothrombinase complex. These results provide new insight into mechanisms through which FXa specifically interacts with its macromolecular substrates in the clotting and signaling pathways.

Carbamazepine mitigates parenteral nutrition-associated liver disease in a novel ambulatory piglet model.

BACKGROUND: Parenteral nutrition (PN) remains a critical therapeutic option in patients who cannot tolerate enteral feeding. However, although lifesaving, PN is associated with significant side effects, including liver injury, the etiology of which is multifactorial. Carbamazepine (CBZ), an antiepileptic medication, is known to modulate hepatic fibrosis and hepatocellular injury in a variety of liver diseases. We hypothesized that CBZ could prevent PN-associated liver disease (PNALD), which we tested by using our novel ambulatory PN piglet model. METHODS: Piglets were fitted with jugular catheters and infusion pumps for PN and randomized to enteral nutrition (n = 7), PN (n = 6), or PN with parenteral CBZ (n = 6) for 2 weeks. Serum and liver tissue were analyzed via light microscopy, quantification of serum liver injury markers, Ki67 and cytokeratin-7 indexing, and real-time quantitative polymerase chain reaction. RESULTS: PN-fed piglets in our model developed manifestations of PNALD-particularly, increased serum bilirubin, gamma-glutamyltransferase, liver cholestasis, and Ki67 expression compared with that of EN-fed animals (P < 0.03). CBZ therapy in PN-fed animals led to a significant reduction in these markers of injury (P < 0.05). Investigation into the mechanism of these therapeutic effects revealed increased expression of sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor alpha (PPAR-α), and fatty acid binding protein (FABP) in PN-fed animals receiving CBZ (P < 0.03). Further investigation revealed increased LC3 expression and decreased lysosomal-associated membrane protein (LAMP1) expression with CBZ (P < 0.03). CONCLUSION: CBZ administration mitigates PNALD severity, suggesting a novel therapeutic strategy targeting PN-associated side effects, and may present a paradigm change to current treatment options.

Role of the residues of the 39-loop in determining the substrate and inhibitor specificity of factor IXa.

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with approximately 40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited approximately 5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with approximately 4-fold decreased k(cat) and approximately 2-fold decreased K(m), although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.

Parenteral Nutrition and Cardiotoxicity.

Parenteral nutrition (PN) is a life-saving nutritional therapy for those situations when patients are unable to receive enteral nutrition. However, despite a multitude of benefits offered by PN, it is associated with a variety of side effects, most notably parenteral nutrition-associated liver disease (PNALD). Adverse effects of PN on other organ systems, such as brain and cardiovascular system, have been poorly studied. There have been several case reports, studies, and a recent animal study highlighting cardiotoxic effects of PN; however, much remains unclear about the underlying mechanisms causing cardiac damage. In this review, we propose a series of potential mechanisms behind PN-associated heart injury, and we provide an overview of therapeutic strategies and recent scientific advances.

Dysbiosis and Intestinal Barrier Dysfunction in Pediatric Congenital Heart Disease Is Exacerbated Following Cardiopulmonary Bypass.

There are no data evaluating the microbiome in congenital heart disease following cardiopulmonary bypass. The authors evaluated patients with congenital heart disease undergoing cardiopulmonary bypass and noncardiac patients undergoing surgery without bypass. Patients with congenital heart disease had differences in baseline microbiome compared with control subjects, and this was exacerbated following surgery with bypass. Markers of barrier dysfunction were similar for both groups at baseline, and surgery with bypass induced significant intestinal barrier dysfunction compared with control subjects. This study offers novel evidence of alterations of the microbiome in congenital heart disease and exacerbation along with intestinal barrier dysfunction following cardiopulmonary bypass.

Inhibitory properties of the P1 Tyr variant of antithrombin.

Antithrombin (AT) and protein Z-dependent protease inhibitor (ZPI) are among two physiological serpin inhibitors in plasma that are involved in the regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has narrower protease specificity, inhibiting only factors Xa (fXa) and XIa. Unlike an Arg at the P1 site of the AT reactive center loop (RCL), this residue is a Tyr in ZPI. To investigate the contribution of P1 Tyr in restricting the specificity of ZPI, we engineered an AT mutant in which the P1 Arg of the RCL was replaced with the P1 Tyr of ZPI (AT-R393Y). The reactivity of AT-R393Y with fXa and thrombin was decreased 155- and 970-fold, respectively. However, the serpin mutant inhibited chymotrypsin with an efficiency higher by >4 orders of magnitude. By contrast, chymotrypsin did not exhibit any reactivity with ZPI. The substitution of Asp-189 of fXa with the corresponding residue of chymotrypsin (Ser) did not improve the reactivity of the protease mutant with AT-R393Y; however, the fXa mutant reacted normally with ZPI. These results suggest that the contribution of P1 Tyr to restricting the protease specificity of ZPI is RCL context-dependent and that in addition to P1 Tyr, other structural features within and/or outside the ZPI RCL are involved in determining the protease specificity of the serpin. The results further suggest that thrombin is less tolerant than fXa in accommodating the nonoptimal P1 Tyr of the AT mutant in its active-site pocket.

Impaired Gut-Systemic Signaling Drives Total Parenteral Nutrition-Associated Injury.

BACKGROUND: Total parenteral nutrition (TPN) provides all nutritional needs intravenously. Although lifesaving, enthusiasm is significantly tempered due to side effects of liver and gut injury, as well as lack of mechanistic understanding into drivers of TPN injury. We hypothesized that the state of luminal nutritional deprivation with TPN drives alterations in gut-systemic signaling, contributing to injury, and tested this hypothesis using our ambulatory TPN model. METHODS: A total of 16 one-week-old piglets were allocated randomly to TPN (n = 8) or enteral nutrition (EN, n = 8) for 3 weeks. Liver, gut, and serum were analyzed. All tests were two-sided, with a significance level of 0.05. RESULTS: TPN resulted in significant hyperbilirubinemia and cholestatic liver injury, p = 0.034. Hepatic inflammation (cluster of differentiation 3 (CD3) immunohistochemistry) was higher with TPN (p = 0.021). No significant differences in alanine aminotransferase (ALT) or bile ductular proliferation were noted. TPN resulted in reduction of muscularis mucosa thickness and marked gut atrophy. Median and interquartile range for gut mass was 0.46 (0.30-0.58) g/cm in EN, and 0.19 (0.11-0.29) g/cm in TPN (p = 0.024). Key gut-systemic signaling regulators, liver farnesoid X receptor (FXR; p = 0.021), liver constitutive androstane receptor (CAR; p = 0.014), gut FXR (p = 0.028), G-coupled bile acid receptor (TGR5) (p = 0.003), epidermal growth factor (EGF; p = 0.016), organic anion transporter (OAT; p = 0.028), Mitogen-activated protein kinases-1 (MAPK1) (p = 0.037), and sodium uptake transporter sodium glucose-linked transporter (SGLT-1; p = 0.010) were significantly downregulated in TPN animals, whereas liver cholesterol 7 alpha-hydroxylase (CyP7A1) was substantially higher with TPN (p = 0.011). CONCLUSION: We report significant alterations in key hepatobiliary receptors driving gut-systemic signaling in a TPN piglet model. This presents a major advancement to our understanding of TPN-associated injury and suggests opportunities for strategic targeting of the gut-systemic axis, specifically, FXR, TGR5, and EGF in developing ameliorative strategies.

Mechanisms of Parenteral Nutrition-Associated Liver and Gut Injury.

Parenteral nutrition (PN) has revolutionized the care of patients with intestinal failure by providing nutrition intravenously. Worldwide, PN remains a standard tool of nutrition delivery in neonatal, pediatric, and adult patients. Though the benefits are evident, patients receiving PN can suffer serious cholestasis due to lack of enteral feeding and sometimes have fatal complications from liver injury and gut atrophy, including PN-associated liver disease or intestinal failure-associated liver disease. Recent studies into gut-systemic cross talk via the bile acid-regulated farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) axis, gut microbial control of the TGR5-glucagon-like peptide (GLP) axis, sepsis, and role of prematurity of hepatobiliary receptors are greatly broadening our understanding of PN-associated injury. It has also been shown that the composition of ω-6/ω-3 polyunsaturated fatty acids given parenterally as lipid emulsions can variably drive damage to hepatocytes and cell integrity. This manuscript reviews the mechanisms for the multifactorial pathogenesis of liver disease and gut injury with PN and discusses novel ameliorative strategies.

Development and validation of an ambulatory piglet model for short bowel syndrome with ileo-colonic anastomosis.

IMPACT STATEMENT: Short bowel syndrome is associated with significant comorbidities and mortality. This study is important as unlike current systems, it provides a validated piglet model which mirrors anatomical, histological, and serological characteristics observed in human SBS. This model can be used to advance knowledge into mechanistic pathways and therapeutic modalities to improve outcomes for SBS patients. This study is novel in that in addition to significant reduction in the remnant bowel and noted liver disease, we also developed a method to emulate ileocecal valve resection and described gut adaptive responses which has important clinical implications in humans.

Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.

Mutagenesis studies toward understanding allostery in thrombin.

The binding of thrombomodulin (TM) to exosite-1 and the binding of Na(+) to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na(+)-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na(+) by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated K(D) to partially restore its activity. Both mutants exhibited approximately 2-3-fold higher K(D) for interaction with Na(+), and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both Na(+)-binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na(+), but TM has no effect on the conformation of this residue.

Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.

Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach (L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 A for APC and 84-86 A for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.