Mechanistically informed predictions of binding modes for carbocation intermediates of a sesquiterpene synthase reaction.

Sesquiterpenoids comprise a class of terpenoid natural products with thousands of compounds that are highly diverse in structure, generally containing a polycyclic carbon backbone that is constructed by a sesquiterpene synthase. Decades of experimental and computational studies have demonstrated that these enzymes generate a carbocation in the active site, which undergoes a series of structural rearrangements until a product is formed via deprotonation or nucleophile attack. However, for the vast majority of these enzymes the productive binding orientation of the intermediate carbocations has remained unclear. In this work, a method that combines quantum mechanics and computational docking is used to generate an all-atom model of every putative intermediate formed in the context of the enzyme active site for tobacco epi-aristolochene synthase (TEAS). This method identifies a single pathway that links the first intermediate to the last, enabling us to propose the first high-resolution model for the reaction intermediates in the active site of TEAS, and providing testable predictions.

Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase.

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

A first-in-human phase I trial of locally delivered human plasmin for hemodialysis graft occlusion.

BACKGROUND: Hemodialysis (HD) grafts often fail because of stenosis at the venous anastomosis and thrombotic occlusion. Percutaneous management relies on thrombolysis with plasminogen activators, mechanical removal of thrombus, and angioplasty of the stenotic lesion. OBJECTIVES: This report describes a phase I trial using Plasmin (Human) TAL 05-00018, a direct-acting fibrinolytic agent, to evaluate safety and, secondarily, to establish effective thrombolytic dosing. PATIENTS/METHODS: Six cohorts of five patients with acute HD graft occlusion documented by angiography were treated with escalating dosages of plasmin (1, 2, 4, 8, 12, and 24 mg) infused over 30 min via criss-crossed pulse-spray catheters within the graft. The primary efficacy endpoint was > or =50% thrombolysis, as determined by comparison of pre-plasmin and 30-min post-plasmin fistulograms. RESULTS: Of 31 subjects who received study drug (safety population), one withdrew and 30 completed the trial (evaluable for efficacy). There was no significant change in plasma alpha-2 antiplasmin or fibrinogen concentration, major bleeding did not occur, and there were no deaths. Serious adverse events in four patients were not related to the study drug. There was a dose-response relationship for the primary efficacy endpoint, all five subjects receiving 24 mg achieving >75% lysis. CONCLUSIONS: This first phase I study of Plasmin (Human) TAL 05-00018, infused into thrombosed HD grafts, documents safety at dosages of 1-24 mg and an effective thrombolytic dosage of 24 mg. The results establish a foundation for further clinical study of catheter-based plasmin administration in thrombotic disorders.

Hydrodynamic thrombectomy system versus pulse-spray thrombolysis for thrombosed hemodialysis grafts: a multicenter prospective randomized comparison.

PURPOSE: To evaluate the safety and efficacy of a hydrodynamic thrombectomy system in a prospective, multicenter randomized comparison with pulse-spray thrombolysis in hemodialysis grafts. MATERIALS AND METHODS: Nine centers enrolled 120 adult patients with recently (

Analysis of CD39/ATP diphosphohydrolase (ATPDase) expression in endothelial cells, platelets and leukocytes.

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ATPDase: mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the coexpressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.

Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity.

CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions" (ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini. ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1, -4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1, -4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.

Disseminated intravascular coagulation in association with the delayed rejection of pig-to-baboon renal xenografts.

BACKGROUND: Intravascular fibrin deposition and platelet sequestration occur with porcine xenograft rejection by baboons. Disseminated intravascular coagulopathy may arise either as a direct consequence of the failure to fully deplete xenoreactive natural antibodies and block complement, or because of putative cross-species molecular incompatibilities in this discordant species combination. METHODS: Three baboons were conditioned with retrovirally transduced autologous bone marrow to induce tolerance to swine antigens. Xenoreactive natural antibodies and complement were depleted by plasmapheresis and the use of Gal alpha1-3Gal column adsorptions; baboons were then splenectomized and underwent renal xenografting from inbred, miniature pigs. Soluble complement receptor type-1 with protocol immunosuppression (mycophenolate mofetil, 15-deoxyspergualin, steroids, and cyclosporine) was administered. RESULTS: A bleeding diathesis was clinically evident from days 5 to 12 after transplantation in two baboons. Low levels of circulating C3a, C3d, and iC3b were measured despite the absence of functional circulating complement components. Profound thrombocytopenia with abnormalities in keeping with disseminated intravascular coagulopathy were observed. Prolongation of prothrombin and partial thromboplastin times was accompanied by evidence for tissue factor-mediated coagulation pathways, high levels of thrombin generation (prothrombin fragment F(1+2) production and thrombin-antithrombin complex formation), fibrinogen depletion, and production of high levels of the fibrin degradation product D-dimer. Importantly, these disturbances resolved rapidly after the excision of the rejected xenografts in two surviving animals. Histopathological examination of the rejected xenografts confirmed vascular injury, fibrin deposition, platelet deposition, and localized complement activation. CONCLUSIONS: Systemic coagulation disturbances are associated with delayed xenograft rejection.

Expression of human thrombomodulin cofactor activity in porcine endothelial cells.

BACKGROUND: Xenograft rejection may predispose to vascular thrombosis because of putative cross-species' functional incompatibilities between natural anticoagulants present on the donor endothelium and host activated coagulation factors. For example, porcine thrombomodulin expressed on porcine aortic endothelial cells (PAEC) does not provide the expected thrombomodulin (TM)-cofactor activity for human protein C in the presence of human thrombin. In addition, TM may be down-regulated after cellular activation. Our aim was to express human TM cofactor activity in PAEC and to study the proinflammatory effect of tumor necrosis factor-alpha (TNF-alpha) on stable expressed human thrombomodulin in vitro. METHODS AND RESULTS: Retroviral transduction of PAEC with the gene encoding for human thrombomodulin (hTM) resulted in expression of high levels of specific TM cofactor activity on PAEC (0.62 microg/ml activated protein C/10(5) cells). High-level expression of hTM resulted in a 620-fold higher activation of human protein C in the presence of human thrombin when compared with mock-transduced PAEC (0.0001 microg/ml/10(5) cells; P<0.001). Transduced PAEC expressing hTM also bound more human thrombin than control PAEC, as determined by inhibition of thrombin-induced platelet activation (P<0.05). We noted that exposure to TNF-alpha significantly reduced exogenous hTM cofactor activity on transduced PAEC in a time- and dose-dependent fashion; this occurred despite the relatively stable expression of hTM mRNA and hTM antigen in these cells. Treatment of transduced PAEC with selected antioxidants could protect against the loss of hTM cofactor activity directly associated with the oxidative stress induced by TNF-alpha activation responses. CONCLUSIONS: Our data show that the functional deficiency of the anticoagulant protein C pathway in PAEC may be corrected by viral transduction of these cells. As analysis of the hTM function showed modulation under conditions of cellular activation, we suggest that expression of hTM mutants resistant to oxidation may have greater therapeutic utility in the genetic modification of porcine xenografts.

Regulation of monocyte tissue factor activity by allogeneic and xenogeneic endothelial cells.

The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNFalpha. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNFalpha-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 +/- 6.3% (p<0.02)] or PAEC [by 50.5 +/- 10.6% (p<0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.

Activation of human platelets by the membrane-expressed A1 domain of von Willebrand factor.

Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF-A1-domains to interact with human platelets in vitro. Both human and porcine vWF-A1-domains expressed as glycosyl phosphatidylinositol-linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.

Xenogeneic endothelial cells activate human prothrombin.

BACKGROUND: Delayed xenograft rejection is characterized by platelet activation and fibrin deposition and is thought to occur independently of complement activation. We have therefore investigated the potential for xenogeneic endothelial cells (EC) to regulate the conversion of prothrombin to thrombin, a central component of the final common pathway of coagulation and an important platelet agonist. METHODS AND RESULTS: Quiescent porcine aortic EC (PAEC) were found to convert high levels of human prothrombin to thrombin (0.234+/-0.019 IU/ml) when compared with human aortic EC (0.017+/-0 IU/ml, 30-min time point, chromogenic assay; P<0.001). PAEC activation by human complement resulted in comparable levels of thrombin generation. Prothrombin conversion by PAEC as determined by generation of F1+2 (1.909+/-0.119 nmol/L) and formation of thrombin-antithrombin III complexes (125.611+/-6.373 microg/L) was significantly greater than the matched human aortic EC values (F1+2: 1.539+/-0.03 nmol/L, P<0.001; thrombin-antithrombin III: 1.833+/-0.104 microg/L, P<0.001). Sequential analysis of prothrombin activation by PAEC indicated generation of the intermediate meizothrombin followed by autolytically accelerated thrombin formation. Subsequent experiments established important cross-species' incompatibilities with respect to porcine thrombomodulin interaction with human thrombin and protein C in that PAEC had a reduced capacity to generate activated human protein C in vitro. CONCLUSION: These observations indicate a potentially important molecular barrier involving blood coagulation that may impact on the planned clinical application of porcine transgenic organs.

Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors.

BACKGROUND: Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. METHODS: In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. RESULTS: Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor alpha. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. CONCLUSIONS: Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.

Modulation of vascular ATP diphosphohydrolase by fatty acids.

Endothelial cells (EC) possess several protective thromboregulatory mechanisms that may be perturbed by cell activation or injury. Vascular ATP-diphosphohydrolase (ATPDase) has been demonstrated on both aortic EC and smooth muscle cells and may play a key regulatory role in hemostasis and platelet reactivity by converting extracellular ATP and ADP to AMP. We have examined the role of exogenous saturated or unsaturated fatty acids in the modulation of EC associated ATPDase activity in vitro. EC growth was not dramatically influenced by supplementation with fatty acids whereas viability was enhanced by oleic, butyrate and eicosapentaenoic acid. EC cultures supplemented with saturated or a monounsaturated (oleic acid) fatty acid(s) had markedly increased ATPDase activity, whereas those exposed to polyunsaturated fatty acids showed substantive decreases. Exogenous oleic acid could also protect against the significant loss of ATPDase activity, following exposure to reactive oxygen intermediates in vitro. We conclude that endothelial ATPDase activity may be regulated by exogenous fatty acids and that underlying mechanisms include alterations in the nature of the phospholipid composition of EC membranes that influence responses to oxidative stress reactions.

Loss of ATP diphosphohydrolase activity with endothelial cell activation.

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

Identification and characterization of CD39/vascular ATP diphosphohydrolase.

Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLGGASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.

Inhibition of T cell mitogenesis by a novel anti-CD45R monoclonal antibody.

CD45 consists of a major family of membrane glycoproteins which have protein tyrosine phosphatase activity and regulate early activation events, progression and maturation signals in leucocytes. Various isoforms of CD45 (Mr 180-240 kDa) regulate sets of intermolecular associations between different surface receptors, and appear to be differentially expressed on B and T cells (namely CD45RA, B or CD45RO). We describe a novel IgG2a mAb directed against restricted and unique CD45R modified epitopes expressed preferentially on peripheral blood T cells. This anti-CD45R antibody (I(2)4c) at concentrations of 50 and 200 ng/mL inhibited mitogenic T cell lectin and anti-CD3-stimulated lymphocyte proliferation and blocked associated IL-2 secretion in vitro. Phorbol ester-stimulated mitogenesis was unaltered suggesting that the inhibition occurs independent of protein kinase C-mediated pathways. Western blotting and immunoprecipitation of purified cell lysates reveals that I(2)4c preferentially binds the higher Mr bands of CD45 expressed on T cells. Following T cell activation in vitro, the 190 kDa band became more predominant and an additional 130 kDa protein, possibly a proteolytic fragment was recognized. I(2)4c may inhibit T cell mitogenesis by direct effects on CD45R alone or by preventing interaction with other membrane-associated proteins and hence adhesive interactions with monocytes. Such interactions may however inhibit the initiation of signal transduction and, as a consequence, alter cellular activation by mitogenic lectins and anti-CD3 in vitro.

The Simon nitinol filter: evaluation by MR and ultrasound.

In this prospective blinded study of inferior vena caval (IVC) patency, 18 patients underwent 25 duplex ultrasound (US) and magnetic resonance (MR) angiography examinations over an eight-month period following Simon nitinol filter placement. Clinical examination for lower extremity venous stasis and plain abdominal radiography were also performed. Twenty-three of 24 MR examinations and 11 of 24 US examinations were judged technically adequate by the blinded observers. One technically adequate US exam was false positive for intraluminal caval thrombus. Thirteen technically inadequate US examinations missed 3 complete caval occlusions and 2 partial occlusions. MR identified all patients with complete or partial caval occlusion. The authors conclude that duplex US reliably confirms IVC patency only when strict criteria for technical adequacy and interpretation are met (good visualization of filter and IVC above and below filter). MR, although expensive, more reliably identifies nonoccluding intraluminal thrombus and caval occlusion. It should be the noninvasive study of choice in symptomatic patients with venous stasis and patients with recurrent pulmonary emboli.

Peripheral directional atherectomy: 4-year experience.

Directional atherectomy alone or with supplemental percutaneous transluminal angioplasty was used to treat peripheral vascular lesions in 77 patients (85 procedures). Lesions involved 17 iliac arteries, 45 infrainguinal arteries, and 23 laser extremity vein bypass grafts. Technical success, defined as reduction of stenosis diameter to 30% or less of the normal vessel diameter, was achieved in 78 of 85 (92%) cases. The complication rate was 21% (18 of 85 procedures). Most complications were minor and were related to puncture sites. Patients underwent noninvasive follow-up studies, including measurement of ankle-brachial index and segmental pressures, plethysmography, and clinical examination. The mean follow-up period was 13.5 months. The probability of 1-, 2-, and 3-year patency for lesions treated with atherectomy alone was 92%, 84%, and 84%, respectively. Kaplan-Meier survival analysis revealed no difference in 2- to 3-year patency rate on the basis of lesion location or presence of calcification, eccentricity, or ulceration. Diabetic patients, however, had a higher restenosis rate than did patients who were not diabetic (P less than .03).