Pivotal Dose of Pembrolizumab: A Dose-Finding Strategy for Immuno-Oncology.

Despite numerous publications emphasizing the value of dose finding, drug development in oncology is dominated by the mindset that higher dose provides higher efficacy. Examples of dose finding implemented by biopharmaceutical firms can change this mindset. The purpose of this article is to outline a pragmatic dose selection strategy for immuno-oncology (IO) and other targeted monoclonal antibodies (mAbs). The approach was implemented for pembrolizumab. Selecting a recommended phase II dose (RP2D) with a novel mechanism of action is often challenging due to uncertain relationships between pharmacodynamics measurements and clinical end points. Additionally, phase I efficacy and safety data are generally inadequate for RP2D selection for IO mAbs. Here, the RP2D was estimated based on phase I (clinical study KN001 A and A2) pharmacokinetics data as the dose required for target saturation, which represents a surrogate for maximal pharmacological effect for antagonist mAbs. Due to limitations associated with collecting and analyzing tumor biopsies, characterizing intratumoral target engagement (TE) is challenging. To overcome this gap, a physiologically-based pharmacokinetic model was implemented to predict intratumoral TE. As tumors are spatially heterogeneous, TE was predicted in well-vascularized and poorly vascularized tumor regions. Additionally, impact of differences in target expression, for example, due to interindividual variability and cancer type, was simulated. Simulations showed that 200 mg every 3 weeks can achieve ≥ 90% TE in clinically relevant scenarios, resulting in the recommendation of 200 mg every 3 weeks as the RP2D. Randomized dose comparison studies (KN001 B2 and D) showing similar efficacy over a fivefold dose/exposure range confirmed the RP2D as the pivotal dose.

Leveraging model-informed approaches for drug discovery and development in the cardiovascular space.

Cardiovascular disease remains a significant global health burden, and development of cardiovascular drugs in the current regulatory environment often demands large and expensive cardiovascular outcome trials. Thus, the use of quantitative pharmacometric approaches which can help enable early Go/No Go decision making, ensure appropriate dose selection, and increase the likelihood of successful clinical trials, have become increasingly important to help reduce the risk of failed cardiovascular outcomes studies. In addition, cardiovascular safety is an important consideration for many drug development programs, whether or not the drug is designed to treat cardiovascular disease; modeling and simulation approaches also have utility in assessing risk in this area. Herein, examples of modeling and simulation applied at various stages of drug development, spanning from the discovery stage through late-stage clinical development, for cardiovascular programs are presented. Examples of how modeling approaches have been utilized in early development programs across various therapeutic areas to help inform strategies to mitigate the risk of cardiovascular-related adverse events, such as QTc prolongation and changes in blood pressure, are also presented. These examples demonstrate how more informed drug development decisions can be enabled by modeling and simulation approaches in the cardiovascular area.

Time dependent pharmacokinetics of pembrolizumab in patients with solid tumor and its correlation with best overall response.

Pembrolizumab is a monoclonal antibody that targets the programmed death-1 receptor to induce immune-mediated clearance (CL) of tumor cells. Originally approved by the US Food and Drug Administration in 2014 for treating patients with unresectable or metastatic melanoma, pembrolizumab is now also used to treat patients with non-small-cell lung cancer, classical Hodgkin lymphoma, head and neck cancer, and urothelial cancer. This paper describes the recently identified feature of pembrolizumab pharmacokinetics, the time-dependent or time-varying CL. Overall results indicate that CL decreases over the treatment period of a typical patient in a pattern well described by a sigmoidal function of time with three parameters: the maximum proportion change in CL from baseline (approximately Imax or exactly eImax - 1), the time to reach Imax/2 (TI50), and a Hill coefficient. Best overall response per response evaluation criteria in solid tumor category was found to be associated with the magnitude of Imax.

Preclinical and translational evaluation of coagulation factor IXa as a novel therapeutic target.

The benefits of novel oral anticoagulants are hampered by bleeding. Since coagulation factor IX (fIX) lies upstream of fX in the coagulation cascade, and intermediate levels have been associated with reduced incidence of thrombotic events, we evaluated the viability of fIXa as an antithrombotic target. We applied translational pharmacokinetics/pharmacodynamics (PK/PD) principles to predict the therapeutic window (TW) associated with a selective small molecule inhibitor (SMi) of fIXa, compound 1 (CPD1, rat fIXa inhibition constant (Ki, 21 nmol/L) relative to clinically relevant exposures of apixaban (rat fXa Ki 4.3 nmol/L). Concentrations encompassing the minimal clinical plasma concentration (C min) of the 5 mg twice daily (BID) dose of apixaban were tested in rat arteriovenous shunt (AVS/thrombosis) and cuticle bleeding time (CBT) models. An I max and a linear model were used to fit clot weight (CW) and CBT. The following differences in biology were observed: (1) antithrombotic activity and bleeding increased in parallel for apixaban, but to a lesser extent for CPD1 and (2) antithrombotic activity occurred at high (>99%) enzyme occupancy (EO) for fXa or moderate (>65% EO) for fIXa. translational PK/PD analysis indicated that noninferiority was observed for concentrations of CPD1 that provided between 86% and 96% EO and that superior TW existed between 86% and 90% EO. These findings were confirmed in a study comparing short interfering (si)RNA-mediated knockdown (KD) modulation of fIX and fX mRNA. In summary, using principles of translational biology to relate preclinical markers of efficacy and safety to clinical doses of apixaban, we found that modulation of fIXa can be superior to apixaban.

Development of a Cucumis sativus TILLinG platform for forward and reverse genetics.

BACKGROUND: Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics. PRINCIPAL FINDINGS: A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation. CONCLUSIONS: We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.

Lipids, safety parameters, and drug concentrations after an additional 2 years of treatment with anacetrapib in the DEFINE study.

Anacetrapib is a cholesteryl ester transfer protein (CETP) inhibitor that has previously been shown to reduce low-density lipoprotein cholesterol (LDL-C) and raise high-density lipoprotein cholesterol (HDL-C) in patients with or at high risk of coronary heart disease in the 76-week, placebo-controlled, Determining the Efficacy and Tolerability of CETP Inhibition with Anacetrapib (DEFINE) trial. Here, we report the results of the 2-year extension to the DEFINE study where patients (n = 803) continued on the same assigned treatment as in the original 76-week study. Treatment with anacetrapib during the 2-year extension was well tolerated with a safety profile similar to patients on placebo. No clinically important abnormalities in liver enzymes, blood pressure, electrolytes, or adverse experiences were observed during the extension. At the end of the extension study, relative to the original baseline value, anacetrapib reduced Friedewald-calculated LDL-C by 39.9% and increased HDL-C by 153.3%, compared to placebo. The apparent steady state mean plasma trough concentration of anacetrapib was ∼640 nmol/L. Geometric mean plasma concentrations of anacetrapib did not appear to increase beyond week 40 of the 2-year extension of the 76-week DEFINE base study. In conclusion, an additional 2 years of treatment with anacetrapib were well tolerated with durable lipid-modifying effects on LDL-C and HDL-C.

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.

BACKGROUND: Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics. RESULTS: A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species. CONCLUSIONS: We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.

Systems biology of platelet-vessel wall interactions.

Blood systems biology seeks to quantify outside-in signaling as platelets respond to numerous external stimuli, typically under flow conditions. Platelets can activate via GPVI collagen receptor and numerous G-protein coupled receptors (GPCRs) responsive to ADP, thromboxane, thrombin, and prostacyclin. A bottom-up ODE approach allowed prediction of platelet calcium and phosphoinositides following P2Y1 activation with ADP, either for a population average or single cell stochastic behavior. The homeostasis assumption (i.e., a resting platelet stays resting until activated) was particularly useful in finding global steady states for these large metabolic networks. Alternatively, a top-down approach involving Pairwise Agonist Scanning (PAS) allowed large data sets of measured calcium mobilization to predict an individual's platelet responses. The data was used to train neural network (NN) models of signaling to predict patient-specific responses to combinatorial stimulation. A kinetic description of platelet signaling then allows prediction of inside-out activation of platelets as they experience the complex biochemical milieu at the site of thrombosis. Multiscale lattice kinetic Monte Carlo (LKMC) utilizes these detailed descriptions of platelet signaling under flow conditions where released soluble species are solved by finite element method and the flow field around the growing thrombus is updated using computational fluid dynamics or lattice Boltzmann method. Since hemodynamic effects are included in a multiscale approach, thrombosis can then be predicted under arterial and venous thrombotic conditions for various anatomical geometries. Such systems biology approaches accommodate the effect of anti-platelet pharmacological intervention where COX1 pathways or ADP signaling are modulated in a patient-specific manner.

The poly(ADP-ribose) polymerase inhibitor niraparib (MK4827) in BRCA mutation carriers and patients with sporadic cancer: a phase 1 dose-escalation trial.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) is implicated in DNA repair and transcription regulation. Niraparib (MK4827) is an oral potent, selective PARP-1 and PARP-2 inhibitor that induces synthetic lethality in preclinical tumour models with loss of BRCA and PTEN function. We investigated the safety, tolerability, maximum tolerated dose, pharmacokinetic and pharmacodynamic profiles, and preliminary antitumour activity of niraparib. METHODS: In a phase 1 dose-escalation study, we enrolled patients with advanced solid tumours at one site in the UK and two sites in the USA. Eligible patients were aged at least 18 years; had a life expectancy of at least 12 weeks; had an Eastern Cooperative Oncology Group performance status of 2 or less; had assessable disease; were not suitable to receive any established treatments; had adequate organ function; and had discontinued any previous anticancer treatments at least 4 weeks previously. In part A, cohorts of three to six patients, enriched for BRCA1 and BRCA2 mutation carriers, received niraparib daily at ten escalating doses from 30 mg to 400 mg in a 21-day cycle to establish the maximum tolerated dose. Dose expansion at the maximum tolerated dose was pursued in 15 patients to confirm tolerability. In part B, we further investigated the maximum tolerated dose in patients with sporadic platinum-resistant high-grade serous ovarian cancer and sporadic prostate cancer. We obtained blood, circulating tumour cells, and optional paired tumour biopsies for pharmacokinetic and pharmacodynamic assessments. Toxic effects were assessed by common toxicity criteria and tumour responses ascribed by Response Evaluation Criteria in Solid Tumors (RECIST). Circulating tumour cells and archival tumour tissue in prostate patients were analysed for exploratory putative predictive biomarkers, such as loss of PTEN expression and ETS rearrangements. This trial is registered with ClinicalTrials.gov, NCT00749502. FINDINGS: Between Sept 15, 2008, and Jan 14, 2011, we enrolled 100 patients: 60 in part A and 40 in part B. 300 mg/day was established as the maximum tolerated dose. Dose-limiting toxic effects reported in the first cycle were grade 3 fatigue (one patient given 30 mg/day), grade 3 pneumonitis (one given 60 mg/day), and grade 4 thrombocytopenia (two given 400 mg/day). Common treatment-related toxic effects were anaemia (48 patients [48%]), nausea (42 [42%]), fatigue (42 [42%]), thrombocytopenia (35 [35%]), anorexia (26 [26%]), neutropenia (24 [24%]), constipation (23 [23%]), and vomiting (20 [20%]), and were predominantly grade 1 or 2. Pharmacokinetics were dose proportional and the mean terminal elimination half-life was 36·4 h (range 32·8-46·0). Pharmacodynamic analyses confirmed PARP inhibition exceeded 50% at doses greater than 80 mg/day and antitumour activity was documented beyond doses of 60 mg/day. Eight (40% [95% CI 19-64]) of 20 BRCA1 or BRCA2 mutation carriers with ovarian cancer had RECIST partial responses, as did two (50% [7-93]) of four mutation carriers with breast cancer. Antitumour activity was also reported in sporadic high-grade serous ovarian cancer, non-small-cell lung cancer, and prostate cancer. We recorded no correlation between loss of PTEN expression or ETS rearrangements and measures of antitumour activity in patients with prostate cancer. INTERPRETATION: A recommended phase 2 dose of 300 mg/day niraparib is well tolerated. Niraparib should be further assessed in inherited and sporadic cancers with homologous recombination DNA repair defects and to target PARP-mediated transcription in cancer. FUNDING: Merck Sharp and Dohme.

SMART--Sunflower Mutant population And Reverse genetic Tool for crop improvement.

BACKGROUND: Sunflower (Helianthus annuus L.) is an important oilseed crop grown widely in various areas of the world. Classical genetic studies have been extensively undertaken for the improvement of this particular oilseed crop. Pertaining to this endeavor, we developed a "chemically induced mutated genetic resource for detecting SNP by TILLING" in sunflower to create new traits. RESULTS: To optimize the EMS mutagenesis, we first conducted a "kill curve" analysis with a range of EMS dose from 0.5% to 3%. Based on the observed germination rate, a 50% survival rate i.e. LD50, treatment with 0.6% EMS for 8 hours was chosen to generate 5,000 M2 populations, out of which, 4,763 M3 plants with fertile seed set. Phenotypic characterization of the 5,000 M2 mutagenised lines were undertaken to assess the mutagenesis quality and to identify traits of interest. In the M2 population, about 1.1% of the plants showed phenotypic variations. The sunflower TILLING platform was setup using Endo-1-nuclease as mismatch detection system coupled with an eight fold DNA pooling strategy. As proof-of-concept, we screened the M2 population for induced mutations in two genes related to fatty acid biosynthesis, FatA an acyl-ACP thioesterase and SAD the stearoyl-ACP desaturase and identified a total of 26 mutations. CONCLUSION: Based on the TILLING of FatA and SAD genes, we calculated the overall mutation rate to one mutation every 480 kb, similar to other report for this crop so far. As sunflower is a plant model for seed oil biosynthesis, we anticipate that the developed genetic resource will be a useful tool to identify novel traits for sunflower crop improvement.

Multiscale prediction of patient-specific platelet function under flow.

During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

Systems biology of coagulation initiation: kinetics of thrombin generation in resting and activated human blood.

Blood function defines bleeding and clotting risks and dictates approaches for clinical intervention. Independent of adding exogenous tissue factor (TF), human blood treated in vitro with corn trypsin inhibitor (CTI, to block Factor XIIa) will generate thrombin after an initiation time (T(i)) of 1 to 2 hours (depending on donor), while activation of platelets with the GPVI-activator convulxin reduces T(i) to ∼20 minutes. Since current kinetic models fail to generate thrombin in the absence of added TF, we implemented a Platelet-Plasma ODE model accounting for: the Hockin-Mann protease reaction network, thrombin-dependent display of platelet phosphatidylserine, VIIa function on activated platelets, XIIa and XIa generation and function, competitive thrombin substrates (fluorogenic detector and fibrinogen), and thrombin consumption during fibrin polymerization. The kinetic model consisting of 76 ordinary differential equations (76 species, 57 reactions, 105 kinetic parameters) predicted the clotting of resting and convulxin-activated human blood as well as predicted T(i) of human blood under 50 different initial conditions that titrated increasing levels of TF, Xa, Va, XIa, IXa, and VIIa. Experiments with combined anti-XI and anti-XII antibodies prevented thrombin production, demonstrating that a leak of XIIa past saturating amounts of CTI (and not "blood-borne TF" alone) was responsible for in vitro initiation without added TF. Clotting was not blocked by antibodies used individually against TF, VII/VIIa, P-selectin, GPIb, protein disulfide isomerase, cathepsin G, nor blocked by the ribosome inhibitor puromycin, the Clk1 kinase inhibitor Tg003, or inhibited VIIa (VIIai). This is the first model to predict the observed behavior of CTI-treated human blood, either resting or stimulated with platelet activators. CTI-treated human blood will clot in vitro due to the combined activity of XIIa and XIa, a process enhanced by platelet activators and which proceeds in the absence of any evidence for kinetically significant blood borne tissue factor.

RGS/Gi2alpha interactions modulate platelet accumulation and thrombus formation at sites of vascular injury.

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I(2) (PGI(2)), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in G(i2α) that blocks RGS/G(i2) interactions to examine the consequences of lifting constraints on G(i2)-dependent signaling without altering receptor:effector coupling. The results show that the G(i2α)(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca(++)](i), Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Pairwise agonist scanning predicts cellular signaling responses to combinatorial stimuli.

Prediction of cellular response to multiple stimuli is central to evaluating patient-specific clinical status and to basic understanding of cell biology. Cross-talk between signaling pathways cannot be predicted by studying them in isolation and the combinatorial complexity of multiple agonists acting together prohibits an exhaustive exploration of the complete experimental space. Here we describe pairwise agonist scanning (PAS), a strategy that trains a neural network model based on measurements of cellular responses to individual and all pairwise combinations of input signals. We apply PAS to predict calcium signaling responses of human platelets in EDTA-treated plasma to six different agonists (ADP, convulxin, U46619, SFLLRN, AYPGKF and PGE(2)) at three concentrations (0.1, 1 and 10 x EC(50)). The model predicted responses to sequentially added agonists, to ternary combinations of agonists and to 45 different combinations of four to six agonists (R = 0.88). Furthermore, we use PAS to distinguish between the phenotypic responses of platelets from ten donors. Training neural networks with pairs of stimuli across the dose-response regime represents an efficient approach for predicting complex signal integration in a patient-specific disease milieu.

A molecular signaling model of platelet phosphoinositide and calcium regulation during homeostasis and P2Y1 activation.

To quantify how various molecular mechanisms are integrated to maintain platelet homeostasis and allow responsiveness to adenosine diphosphate (ADP), we developed a computational model of the human platelet. Existing kinetic information for 77 reactions, 132 fixed kinetic rate constants, and 70 species was combined with electrochemical calculations, measurements of platelet ultrastructure, novel experimental results, and published single-cell data. The model accurately predicted: (1) steady-state resting concentrations for intracellular calcium, inositol 1,4,5-trisphosphate, diacylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol 4,5-bisphosphate; (2) transient increases in intracellular calcium, inositol 1,4,5-trisphosphate, and G(q)-GTP in response to ADP; and (3) the volume of the platelet dense tubular system. A more stringent test of the model involved stochastic simulation of individual platelets, which display an asynchronous calcium spiking behavior in response to ADP. Simulations accurately reproduced the broad frequency distribution of measured spiking events and demonstrated that asynchronous spiking was a consequence of stochastic fluctuations resulting from the small volume of the platelet. The model also provided insights into possible mechanisms of negative-feedback signaling, the relative potency of platelet agonists, and cell-to-cell variation across platelet populations. This integrative approach to platelet biology offers a novel and complementary strategy to traditional reductionist methods.

Determination of surface tissue factor thresholds that trigger coagulation at venous and arterial shear rates: amplification of 100 fM circulating tissue factor requires flow.

Protein microarrays presenting spots of collagen and lipidated tissue factor (TF) allowed a determination of the critical surface concentration of TF required to trigger coagulation under flow. Whole blood supplemented with corn trypsin inhibitor (to inhibit factor XIIa) was perfused over microarrays for 5 minutes. Immunofluorescence staining of platelet glycoprotein GPIbalpha and fibrin(ogen) revealed a critical TF concentration (EC50) of 3.6, 8.4, and 10.2 molecules-TF/microm2 at wall shear rates of 100, 500, and 1000 s(-1), respectively. For collagen arrays where only the center lane of spots (in the direction of flow) contained TF, a downstream distance of 14 mm was required for the thrombus to widen enough to reach across a 300-micrometer gap to the adjacent TF-free lanes of collagen spots, in agreement with numerical simulation. To investigate the effect of low levels of circulating TF, whole blood (+/-100 fM added TF) was tested under static and flow conditions. After 5 minutes, the addition of 100 fM TF to whole blood had negligible effect under static conditions, but caused a 2.5-fold increase in fibrin formation under flow. This report defines the threshold concentrations of surface TF required to trigger coagulation under flow.

Characterisation of disproportionating enzyme from wheat endosperm.

Disproportionating enzyme or D-enzyme (EC 2.4.1.25) is an alpha-1,4 glucanotransferase which catalyses cleavage and transfer reactions involving alpha-1,4 linked glucans altering (disproportionating) the chain length distribution of pools of oligosaccharides. While D-enzyme has been well characterised in some plants, e.g. potato and Arabidopsis, very little is known about its abundance and function in cereals which constitute the major source of starch worldwide. To address this we have investigated D-enzyme in wheat (Triticum aestivum). Two putative D-enzyme cDNA clones have been isolated from tissue-specific cDNA libraries. TaDPE1-e, from an endosperm cDNA library, encodes a putative polypeptide of 575 amino acid residues including a predicted transit peptide of 41 amino acids. The second cDNA clone, TaDPE1-l, from an Aegilops taushii leaf cDNA library, encodes a putative polypeptide of 579 amino acids including a predicted transit peptide of 45 amino acids. The mature polypeptides TaDPE1-e and TaDPE1-l were calculated to be 59 and 60 kDa, respectively, and had 96% identity. The putative polypeptides had significant identity with deduced D-enzyme sequences from corn and rice, and all the expected conserved residues were present. Protein analysis revealed that D-enzyme is present in the amyloplast of developing endosperm and in the germinating seeds. D-enzyme was partially purified from wheat endosperm and shown to exhibit disproportionating activity in vitro by cleaving maltotriose to produce glucose as well as being able to use maltoheptaose as the donor for the addition of glucans to the outer chains of glycogen and amylopectin.

Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves.

A minimum of four soluble starch synthase families have been documented in all starch-storing green plants. These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin. Comparative analysis of beta-limit dextrins for both wild type and mutant amylopectins suggests a specific and crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own characterization of SSI activity and the previously described kinetic properties of maize SSI, our results suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin cluster synthesis.

Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of amylopectin.

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.