Modulation of the activated protein C pathway in severe haemophilia A patients: The effects of thrombomodulin and a factor V-stabilizing fab.

INTRODUCTION: The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APC's inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next-generation therapeutic targets for haemophilia treatment. AIMS: To determine ex vivo the respective concentration-dependent effects of TM and a FV-stabilizing Fab on the APC regulatory pathway in severe FVIII-deficient blood and plasma. METHODS: Ten severe haemophilia A subjects and one healthy control were enrolled. Blood was spiked with TM (0, 1, 2.5, 5, 10, 20.0 nmol/L) and FV-stabilizing Fab (0, 3, 15, 65, 300 nmol/L). The respective effects were compared to FVIII concentrations of 3- and 10% using rotational thromboelastometry clotting time (CT) and thrombin generation analysis (TGA). RESULTS: With 1 and 2.5 nmol/L TM, 5% FVIII resulted in CT similar to the absence of TM, suggesting it completely reversed the effect of APC. Increasing TM concentrations also reduced peak thrombin generation and ETP. The addition of 300 nmol/L FV-stabilizing Fab returned CT to nearly baseline, but for most subjects was less than the effects of 3- or 10% FVIII. The FV-stabilizing Fab produced similar or greater thrombin generation compared to samples with 3- or 10% FVIII. CONCLUSIONS: The FV-stabilizing Fab resulted in enhanced CT and TGA parameters consistent with FVIII levels of 3- and 10%. Additional studies need to further characterize how modulating the APC pathway may prove beneficial in developing new haemophilia drug targets.

Efficacy and safety of point-of-care ultrasound-guided intra-articular corticosteroid joint injections in patients with haemophilic arthropathy.

INTRODUCTION AND OBJECTIVES: Intra-articular corticosteroid injections are standard of care for managing joint pain secondary to osteoarthritis or rheumatoid arthritis but are rarely used in haemophilic arthropathy. We have introduced and evaluated the efficacy and safety of ultrasound-guided corticosteroid injections for pain relief in patients with haemophilic arthropathy. PATIENTS AND METHODS: Ultrasound-guided intra-articular injections performed on haemophilia patients at UCSD between March 2012 and January 2016 were analysed. Needle placement and injection (40 mg triamcinolone; 3-5 mL lidocaine) were performed with musculoskeletal ultrasound and Power Doppler. Analysis included patient demographics, joint-specific parameters such as tissue hypervascularity and effusions, pain relief, and procedure-associated complications. RESULTS: Forty-five injections (14 ankles, 13 elbows, 18 knees) were administered in 25 patients. Advanced arthropathy with hypervascularity and/or effusions was present in 91% and 61% of joints, respectively. Ninety-one per cent of injections resulted in pain relief which was significant in 84% (>30% reduction). Median pain score was reduced from 7 of 10 to 1 of 10 (P < 0.001), usually within 24 h. Median duration of pain relief was 8 weeks (range 1-16 weeks). Haemophilia B patients experienced longer periods of relief, and high Pettersson scores were associated with shorter duration of relief. There were no procedure-associated complications. Repeat ultrasound of eight joints within 4 weeks of injection demonstrated nearly complete resolution of hypervascularity. CONCLUSIONS: Point-of-care ultrasound enabled intra-articular corticosteroid injections that provided highly effective, safe, and relatively long-lasting pain relief in haemophilic arthropathy. This approach should be used to improve pain management in haemophilic arthropathy.

Allosterism-based simultaneous, dual anticoagulant and antiplatelet action: allosteric inhibitor targeting the glycoprotein Ibα-binding and heparin-binding site of thrombin.

BACKGROUND: Allosteric inhibition is a promising approach for developing a new group of anticoagulants with potentially reduced bleeding consequences. Recently, we designed sulfated ß-O4 lignin (SbO4L) as an allosteric inhibitor that targets exosite 2 of thrombin to reduce fibrinogen cleavage through allostery and compete with glycoprotein Ibα to reduce platelet activation. OBJECTIVE: To assess: (i) the antithrombotic potential of a novel approach of simultaneous exosite 2-dependent allosteric inhibition of thrombin and competitive inhibition of platelet activation; and (ii) the promise of SbO4L as the first-in-class antithrombotic agent. METHODS: A combination of whole blood thromboelastography, hemostasis analysis, mouse arterial thrombosis models and mouse tail bleeding studies were used to assess antithrombotic potential. RESULTS AND CONCLUSIONS: SbO4L extended the clot initiation time, and reduced maximal clot strength, platelet contractile force, and the clot elastic modulus, suggesting dual anticoagulant and antiplatelet effects. These effects were comparable to those observed with enoxaparin. A dose of 1 mg of SbO4L per mouse prevented occlusion in 100% of arteries, and lower doses resulted in a proportionally reduced response. Likewise, the time to occlusion increased by ~ 70% with a 0.5-mg dose in the mouse Rose Bengal thrombosis model. Finally, tail bleeding studies demonstrated that SbO4L does not increase bleeding propensity. In comparison, a 0.3-mg dose of enoxaparin increased the bleeding time and blood volume loss. Overall, this study highlights the promise of the allosteric inhibition approach, and presents SbO4L as a novel anticoagulant with potentially reduced bleeding side effects.

Coated platelets and severe haemophilia A bleeding phenotype: Is there a connection?

INTRODUCTION: Coated platelets are a subpopulation of platelets that possess highly prothrombotic properties. Previous observational data suggest that bleeding phenotype in severe haemophilia A is associated with coated platelet levels. Haemophilia A patients with higher coated platelet levels may have a mild bleeding phenotype; those with lower levels may have a more severe bleeding phenotype. AIM: The aim of the study was to test the hypothesis that coated platelet levels are correlated with clinical bleeding phenotype. METHODS: This cross-sectional, observational study enrolled 20 severe haemophilia A patients, including 15 with severe and five with a mild bleeding phenotype, and a control group of 12 healthy volunteers. The haemophilia bleeding phenotype was determined by the patient's medical history and haemophilia treatment centre records. Blood was obtained from each patient by venipuncture and platelets were analysed by flow cytometry. RESULTS: Patients categorized as having a severe bleeding phenotype experienced a median eight bleeds per year compared to one bleed annually in the mild bleeding phenotype group. Both groups had similar total platelet counts and fibrinogen levels. There was no difference in coated platelet percentage between severe and mild bleeding phenotype (17 and 16% respectively), however, both groups had significantly lower % coated platelets compared to controls (44%, P < 0.0001). CONCLUSION: Coated platelet levels were not associated with bleeding phenotype in this study; however, these data may suggest coated platelet levels are lower in haemophilia patients relative to healthy volunteers.

The brain in three crustaceans from cavernous darkness.

BACKGROUND: While a number of neuroanatomical studies in other malacostracan taxa have recently contributed to the reconstruction of the malacostracan ground pattern, little is known about the nervous system in the three enigmatic blind groups of peracarids from relict habitats, Thermosbaenacea, Spelaeogriphacea, and Mictocarididae. This first detailed description of the brain in a representative of each taxon is largely based on a combination of serial semi-thin sectioning and computer-aided 3D-reconstructions. In addition, the mictocaridid Mictocaris halope was studied with a combination of immunolabeling (tubulin, nuclear counter-stains) and confocal laser scanning microscopy, addressing also the ventral nerve cord. RESULTS: Adjacent to the terminal medulla, all three representatives exhibit a distal protocerebral neuropil, which is reminiscent of the lobula in other Malacostraca, but also allows for an alternative interpretation in M. halope and the thermosbaenacean Tethysbaena argentarii. A central complex occurs in all three taxa, most distinctively in the spelaeogriphacean Spelaeogriphus lepidops. The deutocerebral olfactory lobe in M. halope and S. lepidops is large. The comparably smaller olfactory lobe in T. argentarii appears to be associated with a unique additional deutocerebral neuropil. A small hemiellipsoid body exists only in the protocerebrum of T. argentarii. Distinctive mechanosensory neuropils corresponding to other malacostracans are missing. CONCLUSIONS: The considerable reduction of the optic lobe in the studied taxa is higher than in any other blind malacostracan. The large size of deutocerebral olfactory centers implies an important role of the olfactory sense. The presence of a distinctive central complex in the blind S. lepidops adds further support to a central-coordinating over a visual function of this structure. The lack of a hemiellipsoid body in M. halope and S. lepidops suggests that their terminal medulla takes over the function of a second order olfactory center completely, as in some other peracarids. The reduction of the optic lobe and hemiellipsoid body is suggested to have occurred several times independently within Peracarida. The missing optic sense in the studied taxa is not correlated with an emphasized mechanosense.

Early hemostatic responses to trauma identified with hierarchical clustering analysis.

BACKGROUND: Trauma-induced coagulopathy is a complex multifactorial hemostatic response that is poorly understood. OBJECTIVES: To identify distinct hemostatic responses to trauma and identify key components of the hemostatic system that vary between responses. PATIENTS/METHODS: A cross-sectional observational study of adult trauma patients at an urban level I trauma center emergency department was performed. Hierarchical clustering analysis was used to identify distinct clusters of similar subjects according to vital signs, injury/shock severity, and comprehensive assessment of coagulation, clot formation, platelet function, and thrombin generation. RESULTS: Among 84 total trauma patients included in the model, three distinct trauma clusters were identified. Cluster 1 (N = 57) showed platelet activation, preserved peak thrombin generation, plasma coagulation dysfunction, a moderately decreased fibrinogen concentration and normal clot formation relative to healthy controls. Cluster 2 (N = 18) showed platelet activation, preserved peak thrombin generation, and a preserved fibrinogen concentration with normal clot formation. Cluster 3 (N = 9) was the most severely injured and shocked, and showed a strong inflammatory and bleeding phenotype. Platelet dysfunction, thrombin inhibition, plasma coagulation dysfunction and a decreased fibrinogen concentration were present in this cluster. Fibrinolytic activation was present in all clusters, but was particularly increased in cluster 3. Trauma clusters were most noticeably different in their relative fibrinogen concentration, peak thrombin generation, and platelet-induced clot contraction. CONCLUSIONS: Hierarchical clustering analysis identified three distinct hemostatic responses to trauma. Further insights into the underlying hemostatic mechanisms responsible for these responses are needed.

Is some better than none: are TEG and TGA profiles different in severe FVIII-deficient patients with inhibitors?

Severe factor VIII (FVIII)-deficient patients with and without FVIII inhibitors cannot be distinguished using FVIII levels. The FVIII assay is sensitive to detect factor levels below 1%. While severe FVIII-deficient, non-inhibitor patients have FVIII < 1%, they may retain unmeasurable residual factor activity. In contrast, inhibitor patients have a FVIII antibody that presumably fully eliminates FVIII activity. It is unknown whether thromboelastography (TEG) and thrombin generation assay (TGA) can differentiate between patients with FVIII < 1% with and without the presence of FVIII inhibitors. The primary objective was to discern whether TEG and TGA could differentiate between severe FVIII-deficient patients with and without the presence of FVIII inhibitors. A secondary objective was to correlate TEG and TGA to annualized bleeding rates. This observational study performed TEG and TGA in healthy volunteers (N = 15), severe FVIII-deficient (N = 15) and severe FVIII-deficient patients with inhibitors (N = 15). Kaolin-activated TEG was better at differentiating reaction time (31.3 vs. 120 min respectively, P = 0.004) and kinetics time (6.1 vs. 23.1 min respectively, P = 0.028) between the non-inhibitor and inhibitor patients. TEG activated by tissue factor in plasma-containing corn trypsin inhibitor failed to differentiate groups. The TGA failed to differentiate peak thrombin, endogenous thrombin potential and lag time between groups. There was no correlation between TEG and TGA with annualized bleeding rates. Kaolin-activated TEG, but not TGA, differentiated between severe FVIII-deficient patients with and without inhibitors. These assays did not find a correlation to annualized bleeding rate.

Failure of corn trypsin inhibitor to affect the thrombin generation assay in plasma from severe hemophiliacs.

BACKGROUND: The thrombin generation assay (TGA) is an important global coagulation assay; however, it suffers from the lack of preanalytical standardization. The addition of corn trypsin inhibitor (CTI) to blood collection tubes before TGA has been previously advocated to block the contact activation pathway. Emerging data, however, suggest that CTI may only be necessary when minimal tissue factor (TF) concentrations < 1 pmol are used. OBJECTIVES: To determine whether blood collection tubes containing CTI influenced TGA parameters. METHODS: This cross-sectional, observational study performed the TGA using TF 1 pmol L(-1) in 15 healthy volunteers, 14 severely factor VIII (FVIII)-deficient patients, and 15 severely FVIII-deficient patients with documented FVIII inhibitors. TGA was conducted using blood tubes that contained CTI 33 µg mL(-1) and no CTI. RESULTS: CTI markedly reduced peak thrombin (P = 0.002) and endogenous thrombin potential (P < 0.001) in the healthy volunteers but had no significant effect on TGA parameters in severely FVIII-deficient patients or those with inhibitors. CONCLUSIONS: This lack of effect raises additional questions regarding the need for CTI as a preanalytical addition to blood collection tubes during TGA in severe hemophiliacs, particularly when activating samples with TF 1 pmol L(-1) .

The ventral nerve cord in Cephalocarida (Crustacea): new insights into the ground pattern of Tetraconata.

Cephalocarida are Crustacea with many anatomical features that have been interpreted as plesiomorphic with respect to crustaceans or Tetraconata. While the ventral nerve cord (VNC) has been investigated in many other arthropods to address phylogenetic and evolutionary questions, the few studies that exist on the cephalocarid VNC date back 20 years, and data pertaining to neuroactive substances in particular are too sparse for comparison. We reinvestigated the VNC of adult Hutchinsoniella macracantha in detail, combining immunolabeling (tubulin, serotonin, RFamide, histamine) and nuclear stains with confocal laser microscopy, complemented by 3D-reconstructions based on serial semithin sections. The subesophageal ganglion in Cephalocarida comprises three segmental neuromeres (Md, Mx1, Mx2), while a separate ganglion occurs in all thoracic segments and abdominal segments 1-8. Abdominal segments 9 and 10 and the telson are free of ganglia. The maxillar neuromere and the thoracic ganglia correspond closely in their limb innervation pattern, their pattern of mostly four segmental commissures and in displaying up to six individually identified serotonin-like immunoreactive neurons per body side, which exceeds the number found in most other tetraconates. Only two commissures and two serotonin-like immunoreactive neurons per side are present in abdominal ganglia. The stomatogastric nervous system in H. macracantha corresponds to that in other crustaceans and includes, among other structures, a pair of lateral neurite bundles. These innervate the gut as well as various trunk muscles and are, uniquely, linked to the unpaired median neurite bundle. We propose that most features of the cephalocarid ventral nerve cord (VNC) are plesiomorphic with respect to the tetraconate ground pattern. Further, we suggest that this ground pattern includes more serotonin-like neurons than hitherto assumed, and argue that a sister-group relationship between Cephalocarida and Remipedia, as favored by recent molecular analyses, finds no neuroanatomical support.

Monitoring rFVIII prophylaxis dosing using global haemostasis assays.

Secondary factor VIII (FVIII) prophylaxis converts severe haemophiliacs (FVIII:C < 1 IU dL(-1)) to a moderate phenotype (FVIII:C ≥ 1 IU dL(-1)), however, plasma FVIII:C is a poor predictor of bleeding risk. This study used thromboelastography (TEG) and thrombin generation assay (TGA) to quantify coagulation across a 48 h rFVIII prophylaxis period. 10 severe haemophiliacs with varying clinical bleeding phenotypes received their standard rFVIII prophylaxis dose and blood samples were obtained over 48 h. Measured parameters included FVIII:C, TEG and TGA at each time point. FVIII:C pharmacokinetics (PK) and correlation between global assay parameters was performed. The FVIII:C PK parameters were consistent with previous literature. There was significant correlation between FVIII:C and TEG R-time and aPTT (both P < 0.001). Significant correlations existed between FVIII:C and TGA peak, ETP and velocity parameters (all P < 0.001). At 24 h the TEG parameters were sub-therapeutic despite median FVIII:C of 13.0 IU dL(-1). TGA was sensitive to FVIII:C below 1 IU dL(-1). Those with the severest bleeding phenotype had the lowest TGA parameters. There was significant correlation between FVIII:C and TEG and TGA. TEG lost sensitivity at 48 h, but not TGA. Prospective studies are needed to determine whether these data can be used to design individualized rFVIII prophylaxis regimens.

Observation of trapped gas during electrical resistance heating of trichloroethylene under passive venting conditions.

A two-dimensional experiment employing a heterogeneous sand pack incorporating two pools of trichloroethylene (TCE) was performed to assess the efficacy of electrical resistance heating (ERH) under passive venting conditions. Temperature monitoring displayed the existence of a TCE-water co-boiling plateau at 73.4°C, followed by continued heating to 100°C. A 5cm thick gas accumulation formed beneath a fine-grained capillary barrier during and after co-boiling. The capillary barrier did not desaturate during the course of the experiment; the only pathway for gas escape being through perforated wells traversing the barrier. The thickness of the accumulation was dictated by the entry pressure of the perforated well. The theoretical maximum TCE soil concentration within the region of gas accumulation, following gas collapse, was estimated to be 888mg/kg. Post-heating soil sampling revealed TCE concentrations in this region ranging from 27mg/kg to 96.7mg/kg, indicating removal of aqueous and gas phase TCE following co-boiling as a result of subsequent boiling of water. The equilibrium concentrations of TCE in water corresponding to the range of post-treatment concentrations in soil (6.11mg/kg to 136mg/kg) are calculated to range from 19.8mg/l to 440mg/l. The results of this experiment illustrate the importance of providing gas phase venting during the application of ERH in heterogeneous porous media.

Morphology of the brain in Hutchinsoniella macracantha (Cephalocarida, Crustacea).

External morphological features of Cephalocarida have long been interpreted as plesiomorphic with regard to those of other crustaceans. Based on transmission electron microscopy and light microscopy, however, the brain in the cephalocarid Hutchinsoniella macracantha has been shown to contain a number of structures that are more difficult to interpret in an evolutionary context. These include the multi-lobed complex, a unique cluster of neuropils associated with the olfactory lobes. To establish a well-founded comparison of phylogenetically relevant, neuroanatomical data from Cephalocarida to other arthropods, we investigated the brain in H. macracantha using immunolabeling (acetylated α-tubulin, serotonin, RFamide, histamine) and nuclear counter stains of whole mounts and vibratome sections analyzing specimens with confocal laser scanning microscopy and computer-aided 3D-reconstruction. Other 3D-reconstructions were based on serial 1 µm semi-thin sections. The multi-lobed complex features a pedunculus and shows detailed homologies with the mushroom bodies of certain Insecta and Lithobiomorpha (Chilopoda), suggesting that the hemiellipsoid bodies in Remipedia and Malacostraca have derived from a cephalocarid-like pattern. Like the corresponding tracts in Insecta, the olfactory globular tracts linking the multi-lobed complex to the olfactory lobes are ipsilateral, probably constituting the plesiomorphic pattern from which the decussating tracts in Remipedia and Malacostraca have evolved. The olfactory lobes in H. macracantha are uniquely organized into vertical stacks of olfactory glomeruli whose exact shape could not be identified. Similarly to Malacostraca and Insecta, the olfactory glomeruli in H. macracantha are innervated by serotonin-like, RFamide-like, and histamine-like immunoreactive interneurons. This suggests homology of the olfactory lobes across Tetraconata, despite the different morphological organization. Although H. macracantha lacks elongated, unpaired midline neuropils known from the protocerebrum of other Arthropoda, the possible rudiment of a central-body-like neuropil that receives decussating fibers from anterior somata was revealed by the serotonin-like immunoreactive pattern.

Monitoring rFVIIa 90 µg kg⁻¹ dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h.

Recombinant FVIIa is a haemostatic agent administered to patients with severe FVIII or FIX deficiency with inhibitors. Although rFVIIa is effective at stopping bleeding, a reliable assay to monitor its effect is lacking. To characterize the pharmacokinetics and global coagulation effects of rFVIIa for 6 h following a IV dose of 90 µg kg⁻¹. Ten non-bleeding subjects with severe FVIII or FIX deficiency were infused with a single-dose of rFVIIa 90 µg k⁻¹ body weight and blood was collected before and at 0.5, 1, 2, 4 and 6 h postdose. Global haemostasis was characterized throughout the study utilizing whole blood analyses (Hemodyne HAS, TEG, ROTEM). The clearance and half-life of factor FVII:C was estimated as 39.0 ± 8.8 mL h⁻¹ kg⁻¹ and 2.1 ± 0.2 h respectively. There was good inter-assay agreement with respect to clot initiation parameters (R, CT and FOT) and these parameters all fell to a mean of approximately 9 min following rFVIIa dosing. The platelet contractile force (PCF) and clot elastic modulus (CEM) were positively correlated to FVII:C (P < 0.0001), and these parameters were dynamic throughout the 6-h period. The MA and MCF did not correlate to FVII:C nor did they significantly change during the study. Prothrombin F1 + 2 significantly increased following rFVIIa dosing (P < 0.001), but remained steady throughout the study. There was no change in D-dimer concentrations over time. The FOT, R and CT characterized clot initiation following rFVIIa dosing. The PCF and CEM were correlated to FVII:C and characterized the dynamics of platelet function and clot strength over the rFVIIa dosing interval. The clinical significance of these findings needs additional study.

Recombinant human factor VIIa (rFVIIa) cleared principally by antithrombin following intravenous administration in hemophilia patients.

OBJECTIVE: The objective of the present study was to evaluate the pharmacokinetics and the clearance pathways of rFVIIa after intravenous administration to hemophilia patients. METHODS: Ten severe hemophilia patients were included in the study; all patients were intravenously administered a clinically relevant dose of 90 µg kg(-1) (1.8 nmol kg(-1)) rFVIIa. Blood samples were collected consecutively to describe the pharmacokinetics of rFVIIa. All samples were analyzed using three different assays: a clot assay to measure the activity (FVIIa:C), an enzyme immunoassay (EIA) to measure the antigen levels (FVII:Ag), and an EIA (FVIIa-AT) to measure the FVIIa antithrombin III (AT) complex. Pharmacokinetic parameters were evaluated both by use of standard non-compartmental methods and by use of mixed effects methods. A population pharmacokinetic model was used to simultaneously model all three datasets. The total body clearance of rFVIIa:C was estimated to be 38 mL h(-1) kg(-1). The rFVII-AT complex formation was responsible for 65% of the total rFVIIa:C clearance. The initial and the terminal half-life of rFVIIa:C was estimated to be 0.6 and 2.6 h, respectively. The formation of rFVII-AT complex was able to explain the difference observed between the rFVIIa:C and the rFVII:Ag concentration. The non-compartmental analysis resulted in almost identical parameters.

Effect of recombinant factor VIIa variant (NN1731) on platelet function, clot structure and force onset time in whole blood from healthy volunteers and haemophilia patients.

NN1731 is a novel variant of recombinant factor VIIa (rFVIIa) that binds to activated platelets, but has greater enzymatic activity than rFVIIa in generating FXa and thrombin. The effect of NN1731 on clot structure and platelet function was characterized ex vivo in whole blood from healthy volunteers and haemophilic patients. Blood samples from six healthy volunteers, nine haemophilia A patients with and without inhibitors and one acquired haemophilia A patient, were spiked with increasing concentrations (0.32, 0.64 and 1.28 microg mL(-1)) of rFVIIa and NN1731. Platelet contractile force (PCF) or platelet function, clot elastic modulus (CEM) or clot structure, and force onset time (FOT) or the thrombin generation time (TGT) were determined using the Hemodyne Hemostasis Analysis System (HAS). Baseline PCF, CEM and FOT values in patients were abnormal compared to healthy volunteers' baseline values. Overall, haemophilia blood samples with or without inhibitors spiked with NN1731 had significantly greater PCF, CEM and shorter FOT values relative to samples spiked with corresponding doses of rFVIIa. The variability in response to treatment between patients was greater with rFVIIa compared to NN1731. At 1.28 microg mL(-1) (90 microg kg(-1)), NN1731 normalized PCF, CEM and FOT in nine of 10 patients, while rFVIIa normalized these parameters in four of 10 patients. Increasing in vitro concentrations of NN1731 normalized platelet function, clot structure and thrombin generation consistently in haemophilia blood with or without inhibitors. NN1731 may be a promising haemostatic agent for patients with bleeding disorders. These results should be confirmed in an in vivo study.

The Separative Bioreactor: A Continuous Separation Process for the Simultaneous Production and Direct Capture of Organic Acids.

The replacement of petrochemicals with biobased chemicals requires efficient bioprocesses, biocatalysis, and product recovery. Biocatalysis (e.g., enzyme conversion and fermentation) offers an attractive alternative to chemical processing because biocatalysis utilize renewable feedstocks under benign reaction conditions. One class of chemical products that could be produced in large volumes by biocatalysis is organic acids. However, biocatalytic reactions to produce organic acids typically result in only dilute concentrations of the product because of product inhibition and acidification that drives the reaction pH outside of the optimal range for the biocatalyst. Buffering or neutralization results in formation of the acid salt rather than the acid, which requires further processing to recover the free acid product. To address these barriers to biocatalytic organic acid production, we developed the "separative bioreactor" based on resin wafer electrodeionization, which is an electro-deionization platform that uses resin wafers fabricated from ion exchange resins. The separative bioreactor simultaneously separates the organic acid from the biocatalyst as it is produced, thus it avoids product inhibition enhancing reaction rates. In addition, the separative bioreactor recovers the product in its acid form to avoid neutralization. The instantaneous separation of acid upon formation in the separative bioreactor is one of the first truly one-step systems for producing organic acids. The separative bioreactor was demonstrated with two systems. In the first demonstration, the enzyme glucose fructose oxidoreductase (GFOR) was immobilized in the reactor and later regenerated in situ. GFOR produced gluconic acid (in its acid form) continuously for 7 days with production rates up to 1000 mg/L/hr at >99% product recovery and GFOR reactivity >30mg gluconic acid/mg GFOR/hour. In the second demonstration, the E. coli strain CSM1 produced lactic acid for up to 24 hours with a productivity of >200 mg/L/hr and almost 100% product recovery.

Thrombin generation time is a novel parameter for monitoring enoxaparin therapy in patients with end-stage renal disease.

BACKGROUND: Patients with end-stage renal disease (ESRD) who receive enoxaparin are at increased risk for adverse bleeding episodes. This phenomenon appears to occur despite judicious monitoring of antifactor Xa (aFXa) activity. Better monitoring parameters are needed to quantify the anticoagulant effects of enoxaparin in the ESRD population. OBJECTIVES: The objective of this study was to determine the utility of using thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM) to monitor the degree of anticoagulation in ESRD subjects, and to compare these results to aFXa activity, the current gold-standard monitoring parameter. METHODS: Eight healthy volunteers without renal dysfunction and eight ESRD subjects were enrolled into this study. Subjects received a single dose of enoxaparin 1 mg kg(-1) subcutaneously, and blood samples were obtained for the determination of aFXa activity, TGT, PCF and CEM at baseline, 4, 8, and 12 h postdose. RESULTS: Baseline, 4, 8, and 12-h aFXa activity concentrations were not different between groups. However, the corresponding TGT at 8 and 12 h was significantly prolonged in the ESRD group (P = 0.04, and P = 0.008, respectively). The 4-h peak TGT trended toward significance (P = 0.06). There were no differences in PCF or CEM across time. CONCLUSIONS: These data suggest that the parameter aFXa activity is a poor predictor of the anticoagulant effect of enoxaparin in patients with ESRD. Thrombin generation time appears to be more sensitive to the antithrombotic effects of enoxaparin in this population. Further large-scale trials are needed to corroborate these data.

Antifactor Xa activity correlates to thrombin generation time, platelet contractile force and clot elastic modulus following ex vivo enoxaparin exposure in patients with and without renal dysfunction.

Antifactor Xa activity is the gold standard monitoring parameter for low molecular weight heparin (LMWH) derivatives. It is frequently measured in high-risk populations, such as patients with renal dysfunction. Despite antifactor Xa monitoring, however, bleeding in renal dysfunction patients receiving LMWH remains a problem. This study determined the relationship between antifactor Xa activity and three novel coagulation monitoring parameters: thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM). This study also assessed the effect of renal dysfunction on these relationships. This was an ex vivo pharmacodynamic study of the relationship between antifactor Xa activity and TGT, PCF and CEM in subjects both with and without renal dysfunction. Thirty subjects completed this study (10 controls, 10 chronic kidney disease subjects, and 10 end-stage renal disease subjects receiving hemodialysis). Blood samples obtained from participants were spiked with increasing enoxaparin concentrations (0.25, 0.5, 1.0 and 3.0 IU mL(-1)). Samples were analyzed for TGT, PCF and CEM. The relationship between antifactor Xa activity and TGT, PCF and CEM was determined by Pearson's correlation. The effect of renal dysfunction on the relationship between antifactor Xa activity and TGT, PCF and CEM was determined by analysis of covariance. There is strong correlation between antifactor Xa activity and TGT, CEM and PCF. The presence of renal dysfunction significantly prolongs the TGT, and decreases the CEM relative to controls. These results suggest that patients with renal dysfunction have a greater pharmacodynamic response to LMWH, independent of the pharmacokinetics of LMWH.

Onset of force development as a marker of thrombin generation in whole blood: the thrombin generation time (TGT).

Prothrombin activation requires the direct interplay of activated platelets and plasma clotting factors. Once formed, thrombin causes profound, irreversible activation of platelets and reinforces the platelet plug via fibrin formation. Delayed or deficient thrombin production increases bleeding risk. Commonly employed coagulation assays, the prothrombin and partial thromboplastin times, use clot formation as a surrogate marker of thrombin generation. These assays routinely utilize platelet-poor plasma and completely miss the effects of platelets. Other markers of thrombin generation, prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex, are typically measured after the fact. We report a simple assay, which employs the onset of platelet contractile force (PCF) as a surrogate marker of thrombin generation. PCF generation occurs concomitant with the burst of F1 + 2 release. The time between assay start and PCF onset is termed the thrombin generation time (TGT). TGT is prolonged in clotting factor deficiencies and in the presence of direct and indirect thrombin inhibitors. TGT shortens to normal with clotting factor replacement and shortens with administration of recombinant factor VIIa. TGT is short in thrombophilic states such as coronary artery disease, diabetes and thromboangiitis obliterans and prolongs toward normal with oral and intravenous anticoagulants.