bluuebunny/crossref_metadata_2025 huggingface dataset:
Marinobufagenin enhances cardiac contractility in mice with ouabain-sensitive α1Na+-K+-ATPase
Authors: Wansapura Arshani N. , Lasko Valerie , Xie Zijian , Fedorova Olga V. , Bagrov Alexei Y. , Lingrel Jerry B , Lorenz John N.
Published: April 2009

Endogenous Na+pump inhibitors are thought to play important (patho)physiological roles and occur in two different chemical forms in the mammalian circulation: cardenolides, such as ouabain, and bufadienolides, such as marinobufagenin (MBG). Although all α Na+-K+-ATPase isoforms (α1-4) are sensitive to ouabain in most species, in rats and mice the ubiquitously expressed α1Na+-K+-ATPase is resistant to ouabain. We have previously shown that selective modification of the putative ouabain binding site of either the α1or α2Na+-K+-ATPase subunit in mice substantially alters the cardiotonic influence of exogenously applied cardenolides. To determine whether the ouabain binding site also interacts with MBG and if this interaction plays a functional role, we evaluated cardiovascular function in α1-resistant/α2-resistant (α1R/Rα2R/R), α1-sensitive/α2-resistant (α1S/Sα2R/R), and α1-resistant/α2-sensitive mice (α1R/Rα2S/S, wild type). Cardiovascular indexes were evaluated in vivo by cardiac catheterization at baseline and during graded infusions of MBG. There were no differences in baseline measurements of targeted mice, indicating normal hemodynamics and cardiac function. MBG at 0.025, 0.05, and 0.1 nmol·min−1·g body wt−1significantly increased cardiac performance to a greater extent in α1S/Sα2R/Rcompared with α1R/Rα2R/Rand wild-type mice. The increase in LVdP/d tmaxin α1S/Sα2R/Rmice was greater at higher concentrations of MBG compared with both α1R/Rα2R/Rand α1R/Rα2S/Smice ( P < 0.05). These results suggest that MBG interacts with the ouabain binding site of the α1Na+-K+-ATPase subunit and can thereby influence cardiac inotropy.
AMP-activated protein kinase influences metabolic remodeling in H9c2 cells hypertrophied by arginine vasopressin
Authors: Saeedi Ramesh , Saran Varun V. , Wu Sherry S. Y. , Kume Erika S. , Paulson Kim , Chan Annie P. K. , Parsons Hannah L. , Wambolt Richard B. , Dyck Jason R. B. , Brownsey Roger W. , Allard Michael F.
Published: April 2009

Substrate use switches from fatty acids toward glucose in pressure overload-induced cardiac hypertrophy with an acceleration of glycolysis being characteristic. The activation of AMP-activated protein kinase (AMPK) observed in hypertrophied hearts provides one potential mechanism for the acceleration of glycolysis. Here, we directly tested the hypothesis that AMPK causes the acceleration of glycolysis in hypertrophied heart muscle cells. The H9c2 cell line, derived from the embryonic rat heart, was treated with arginine vasopressin (AVP; 1 μM) to induce a cellular model of hypertrophy. Rates of glycolysis and oxidation of glucose and palmitate were measured in nonhypertrophied and hypertrophied H9c2 cells, and the effects of inhibition of AMPK were determined. AMPK activity was inhibited by 6-[4-(2-piperidin-1- yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo-[1,5-a]pyrimidine (compound C) or by adenovirus-mediated transfer of dominant negative AMPK. Compared with nonhypertrophied cells, glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation in hypertrophied cells, a metabolic profile similar to that of intact hypertrophied hearts. Inhibition of AMPK resulted in the partial reduction of glycolysis in AVP-treated hypertrophied H9c2 cells. Acute exposure of H9c2 cells to AVP also activated AMPK and accelerated glycolysis. These elevated rates of glycolysis were not altered by AMPK inhibition but were blocked by agents that interfere with Ca2+signaling, including extracellular EGTA, dantrolene, and 2-aminoethoxydiphenyl borate. We conclude that the acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK, whereas the acute glycolytic effects of AVP are AMPK independent and at least partially Ca2+dependent.
Novel critical role of Toll-like receptor 4 in lung ischemia-reperfusion injury and edema
Authors: Zanotti Giorgio , Casiraghi Monica , Abano John B. , Tatreau Jason R. , Sevala Mayura , Berlin Hilary , Smyth Susan , Funkhouser William K. , Burridge Keith , Randell Scott H. , Egan Thomas M.
Published: April 2009

Toll-like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes. We employed a murine model of hilar clamping (1 h) with reperfusion times between 15 min and 3 h in TLR4-sufficient (C3H/OuJ) and TLR4-deficient (C3H/HeJ) anesthetized mice with additional studies in chimeric and myeloid differentiation factor 88 (MyD88)- and TLR4-deficient mice to determine the role of TLR4 in lung ischemia-reperfusion injury. Human pulmonary microvascular endothelial monolayers were subjected to simulated warm ischemia and reperfusion with and without CRX-526, a competitive TLR4 inhibitor. Functional TLR4 solely on pulmonary parenchymal cells, not bone marrow-derived cells, mediates early lung edema following ischemia-reperfusion independent of MyD88. Activation of MAPKs and NF-κB was significantly blunted and/or delayed in lungs of TLR4-deficient mice as a consequence of ischemia-reperfusion injury, but edema development appeared to be independent of activation of these signaling pathways. Pretreatment with a competitive TLR4 inhibitor prevented edema in vivo and reduced actin cytoskeletal rearrangement and gap formation in pulmonary microvascular endothelial monolayers subjected to simulated warm ischemia and reperfusion. In addition to its well-accepted role to alter gene transcription, functioning TLR4 on pulmonary parenchymal cells plays a key role in very early and profound pulmonary edema in murine lung ischemia-reperfusion injury. This may be due to a novel mechanism: regulation of endothelial cell cytoskeleton affecting microvascular endothelial cell permeability.
High-fat feeding increases insulin receptor and IRS-1 coimmunoprecipitation with SOCS-3, IKKα/β phosphorylation and decreases PI-3 kinase activity in muscle
Authors: Yaspelkis Ben B. , Kvasha Ilya A. , Figueroa Tania Y.
Published: April 2009

Suppressor of cytokine signaling (SOCS) proteins and/or activation of the proinflammatory pathway have been postulated as possible mechanisms that may contribute to skeletal muscle insulin resistance. Thus, the aims of the present investigation were to determine in high-fat-fed skeletal muscle: 1) whether SOCS-3 protein concentration is increased, 2) whether coimmunoprecipitation of SOCS-3 with the insulin receptor-β subunit and/or IRS-1 is increased, and 3) whether select components of the proinflammatory pathway are altered. Thirty-two male Sprague-Dawley rats were assigned to either control (CON, n = 16) or high-fat-fed (HF, n = 16) dietary groups for 12 wk and then subjected to hind limb perfusions in the presence ( n = 8/group) or absence ( n = 8/group) of insulin. Insulin-stimulated skeletal muscle 3-MG transport rates and PI-3 kinase activity were greater ( P < 0.05) in CON. IRS-1 tyrosine phosphorylation was decreased ( P < 0.05), and IRS-1 serine 307 phosphorylation was increased ( P < 0.05) in HF. Insulin receptor-β (IR-β) subunit coimmunoprecipitation with IRS-1 was reduced in HF. SOCS-3 protein concentration and SOCS-3 coimmunoprecipitation with both the IR-β subunit and IRS-1 was increased ( P < 0.05) in HF. IKKα/β serine phosphorylation was increased ( P < 0.05), IκBα protein concentration was decreased ( P < 0.05) and IκBα serine phosphorylation was increased ( P < 0.05) in HF. Increased colocalization of SOCS-3 with both the IR-β subunit and IRS-1 may provide steric hindrance that prevents IRS-1 from interacting with IR-β, while increased IKKβ serine phosphorylation may contribute to increasing IRS-1 serine phosphorylation, both of which independently can have deleterious effects on insulin-stimulated PI-3 kinase activation in high-fat-fed rodent skeletal muscle.
Wireless Neural Stimulation in Freely Behaving Small Animals
Authors: Arfin Scott K. , Long Michael A. , Fee Michale S. , Sarpeshkar Rahul
Published: April 2009

We introduce a novel wireless, low-power neural stimulation system for use in freely behaving animals. The system consists of an external transmitter and a miniature, implantable wireless receiver–stimulator. The implant uses a custom integrated chip to deliver biphasic current pulses to four addressable bipolar electrodes at 32 selectable current levels (10 μA to 1 mA). To achieve maximal battery life, the chip enters a sleep mode when not needed and can be awakened remotely when required. To test our device, we implanted bipolar stimulating electrodes into the songbird motor nucleus HVC (formerly called the high vocal center) of zebra finches. Single-neuron recordings revealed that wireless stimulation of HVC led to a strong increase of spiking activity in its downstream target, the robust nucleus of the arcopallium. When we used this device to deliver biphasic pulses of current randomly during singing, singing activity was prematurely terminated in all birds tested. Thus our device is highly effective for remotely modulating a neural circuit and its corresponding behavior in an untethered, freely behaving animal.
Cholecystokinin Excites Interneurons in Rat Basolateral Amygdala
Authors: Chung Leeyup , Moore Scott D.
Published: April 2009

The amygdala formation is implicated in generation of emotional states such as anxiety and fear. Many substances that modulate neuronal activity in the amygdala alter anxiety. Cholecystokinin (CCK) is an endogenous neuropeptide that induces anxiety states in behavioral studies in both animals and humans. Using a brain slice preparation, we found that application of CCK increases inhibitory synaptic transmission measured in projection neurons of the basolateral amygdala. To determine the source of the increased inhibition we examined the direct effect of CCK on local interneurons in this region. CCK most strongly depolarized fast-spiking interneurons. Burst-firing and regular-firing interneurons were also depolarized, although to a lesser degree. However, another distinct group of interneurons was unaffected by CCK. These effects were mediated by the CCKBreceptor subtype. The excitatory effect of CCK appeared to be mediated by both a nonselective cation and a K+current.
Task-Dependent Modulation of Primary Afferent Depolarization in Cervical Spinal Cord of Monkeys Performing an Instructed Delay Task
Authors: Seki Kazuhiko , Perlmutter Steve I. , Fetz Eberhard E.
Published: April 2009

Task-dependent modulation of primary afferent depolarization (PAD) was studied in the cervical spinal cord of two monkeys performing a wrist flexion and extension task with an instructed delay period. We implanted two nerve cuff electrodes on proximal and distal parts of the superficial radial nerve (SR) and a recording chamber over a hemi-laminectomy in the lower cervical vertebrae. Antidromic volleys (ADVs) in the SR were evoked by intraspinal microstimuli (ISMS, 3–10 Hz, 3–30 μA) applied through a tungsten microelectrode, and the area of each ADV was measured. In total, 434 ADVs were evoked by ISMS in two monkeys, with onset latency consistently shorter in the proximal than distal cuffs. Estimated conduction velocity suggest that most ADVs were caused by action potentials in cutaneous fibers originating from low-threshold tactile receptors. Modulation of the size of ADVs as a function of the task was examined in 281 ADVs induced by ISMS applied at 78 different intraspinal sites. The ADVs were significantly facilitated during active movement in both flexion and extension ( P < 0.05), suggesting an epoch-dependent modulation of PAD. This facilitation started 400–900 ms before the onset of EMG activity. Such pre-EMG modulation is hard to explain by movement-induced reafference and probably is associated with descending motor commands.
Loss of Potassium Homeostasis Underlies Hyperthermic Conduction Failure in Control and Preconditioned Locusts
Authors: Money Tomas G. A. , Rodgers Corinne I. , McGregor Stuart M. K. , Robertson R. Meldrum
Published: April 2009

At extreme temperature, neurons cease to function appropriately. Prior exposure to a heat stress (heat shock [HS]) can extend the temperature range for action potential conduction in the axon, but how this occurs is not well understood. Here we use electrophysiological recordings from the axon of a locust visual interneuron, the descending contralateral movement detector (DCMD), to examine what physiological changes result in conduction failure and what modifications allow for the observed plasticity following HS. We show that at high temperature, conduction failure in the DCMD occurred preferentially where the axon passes through the thoracic ganglia rather than in the connective. Although the membrane potential hyperpolarized with increasing temperature, we observed a modest depolarization (3–6 mV) in the period preceding the failure. Prior to the conduction block, action potential amplitude decreased and half-width increased. Both of these failure-associated effects were attenuated following HS. Extracellular potassium concentration ([K+]o) increased sharply at failure and the failure event could be mimicked by the application of high [K+]o. Surges in [K+]owere muted following HS, suggesting that HS may act to stabilize ion distribution. Indeed, experimentally increased [K+]olowered failure temperature significantly more in control animals than in HS animals and experimentally maintained [K+]owas found to be protective. We suggest that the more attenuated effects of failure on the membrane properties of the DCMD axon in HS animals is consistent with a decrease in the disruptive nature of the [K+]o-dependent failure event following HS and thus represents an adaptive mechanism to cope with thermal stress.
A Population-Based Clinical Trial of Irinotecan and Carboplatin
Authors: Lau Derick , Huynh Minh , Johl Jewel
Published: April 2009

Purpose. Phase I trials of anticancer drugs are commonly conducted using the method of modified Fibonacci. We have developed a population-based design for phase I trials of combining anticancer drugs such as irinotecan and carboplatin.Patients and Methods. Intrapatient dose escalation of irinotecan and carboplatin was performed according to a predetermined schema to reach individual dose-limiting toxicity (DLT) in 50 patients with solid tumors refractory to previous chemotherapy. The individual toxicity-limiting dose levels were analyzed for normal distribution using the method of Ryan-Joiner and subsequently used to determine a population-based maximum tolerated dose (pMTD). For comparison, a simulation study was performed using the method of modified Fibonacci.Results. The most common dose-limiting toxicities (DLTs) included neutropenia (58%), thrombocytopenia (16%), and diarrhea (8%). The frequency of individual toxicity-limiting dose levels of 50 patients approximated a normal distribution. The dose levels associated with individual limiting toxicities ranged from level 1 (irinotecan 100 mg/m2and carboplatin AUC = 4 mg/mLxmin) to level 8 (irinotecan 350 mg/m2and carboplatin AUC = 6). The pMTD was determined to be dose level 3 (150 mg/m2for irinotecan and AUC = 5 for carboplatin). In contrast, the MTD was determined to be dose level 4 (200 mg/m2for irinotecan and AUC 5 for carboplatin) by modified-Fibonacci simulation.Conclusions. The population-based design of phase I trial allows optimization of dose intensity and derivation of a pMTD. The pMTD has been applied in phase II trial of irinotecan and carboplatin in patients with small-cell lung cancer.
Multiband Antennas for SDR Applications
Authors: Surducan E. , Surducan V. , Iancu D. , Glossner J.
Published: April 2009

We present multiband antennas configurations for SDR applications. Using a composite folded dipole structure as starting point, we derived more complex antenna configurations to support multiple communication protocols for mobile application with linear and circular polarizations. Prototypes as single antenna with circular polarization, tunable single antenna with PIN diode and MIMO systems with three and four antennas, all derivatives of the same basic structure, were produced in an iterative fashion until the desired parameters were achieved. These antennas are suitable for microstrip circuit realizations and can be included in the printed circuit board (PCB) of the device, or used as stand alone. The shapes and measurement results are presented throughout the paper. From the illustrated graphs it can be seen that the stand-alone antennas exhibit positive gain for all the frequency bands of interest while the separation between antennas, for the multiple-input multiple-output (MIMO) case, is better than 15 dB.
MBX‐102/JNJ39659100, a Novel Non‐TZD Selective Partial PPAR‐γ Agonist Lowers Triglyceride Independently of PPAR‐α Activation
Authors: Chandalia Apurva , Clarke Holly J. , Clemens L. Edward , Pandey Bindu , Vicena Vic , Lee Paul , Lavan Brian E. , Gregoire Francine M.
Published: April 2009

MBX‐102/JNJ‐39659100 (MBX‐102) is a selective, partial PPAR‐γ agonist that lowers glucose in the absence of some of the side effects, such as weight gain and edema, that are observed with the TZDs. Interestingly MBX‐102 also displays pronounced triglyceride lowering in preclinical rodent models and in humans. Although in vitro reporter gene studies indicated that MBX‐102 acid is a highly selective PPAR‐γ agonist that lacks PPAR‐α activity, we sought to determine if PPAR‐α activation in vivo could possibly contribute to the triglyceride lowering abilities of MBX‐102. In vivo studies using ZDF and ZF rats demonstrated that MBX‐102 lowered plasma triglycerides. However in ZF rats, MBX‐102 had no effect on liver weight or on hepatic expression levels of PPAR‐α target genes. Further in vitro studies in primary human hepatocytes supported these findings. Finally, the ability of MBX‐102 to lower triglycerides was maintained in PPAR‐α knockout mice, unambiguously establishing that the triglyceride lowering effect of MBX‐102 is PPAR‐α independent. The in vivo lipid lowering abilities of MBX‐102 are therefore mediated by an alternate mechanism which is yet to be determined.
Simple Use of the Suppository Type Povidone-Iodine Can Prevent Infectious Complications in Transrectal Ultrasound-Guided Prostate Biopsy
Authors: Park Dong Soo , Oh Jong Jin , Lee Jin Ha , Jang Woong Ki , Hong Young Kwon , Hong Sung Keun
Published: April 2009

Purpose. To determine the effect of simple use of suppository povidone-iodine on infectious complications after transrectal ultrasonography-guided biopsy of the prostate.Methods. All 481 patients are included and received antibiotic prophylaxis. Among them, 360 patients received povidone-iodine suppository (Gynobetadine; 200 mg) immediately prior to biopsy and 121 patients did not. Infectious complications were classified. To evaluate bactericidal effects, we counted bacterial colonies in the rectum, harvested from a rectal swab before insertion of the suppository and after biopsy. Aliquots of the suspended bacterial strains were added to Mueller-Hinton agar medium for incubation. Colony counts were determined.Results. Infectious complications developed in 1 case (0.3%) in the rectal preparation group (Group 1) and in 8 cases (6.6%) in the nonrectal preparation group (Group 2). One in Group 1 had a fever without sepsis. Two patients had sepsis and six had fever without sepsis in Group 2. Rectal preparation was a statistically significant risk factor influencing the development of infectious complications. In vitro experiments, the mean number of colony-forming units decreased 99.9% after the rectal povidone-iodine preparation.Conclusions. All through the biopsy, povidone-iodine melted into the rectum and decreased the bacterial colony count. Simple use of povidone-iodine suppository before prostate biopsy minimizes the risk of infectious complications.
Kinetic Analyses of Potentiation of Plasminogen Activation by Streptokinase in the Presence of Fibrin or Its Degradation Products
Authors: Takada Yumiko , Takada Akikazu
Published: April 2009

When Glu-plasminogen (Glu-plg) was activated by various amounts of streptokinase (SK), Km of the hydrolysis of S-2251 by the mixture of Glu-plg and SK did not change, but Vmax increased with an increase in the amount of SK. Since low concentrations of SK-plg complex do not result in its conversion to the SK-plasmin complex by mutual activation, these results seem to suggest that the SK-plg complex may be a better activator when S-2251 is used as a substrate. When Glu-plg was activated by SK in the presence of fibrin(ogen) or its degradation products (potentiating agents), Km did not change with an increase in the concentration of each potentiating agent, but Vmax increased. The effects of potentiating agents on the kcat values of these activators were in the order of fibrin > fibrinogen > D > E.
High-Dose Systemic Streptokinase and Acylated Streptokinase-Plasminogen Complex (BRL 26921) in Acute Myocardial Infarction: Alterations of the Fibrinolytic System and Clearance of Fibrinolytic Activity
Authors: Köhler M. , Hellstern P. , Doenecke P. , Schwerdt H. , Özbek C. , Miyashita C. , Winter R.W. , von Blohn G. , Bette L. , Wenzel E.
Published: April 2009

We report the results of two consecutive studies using intravenous bolus injections of streptokinase (SK) or acylated plasminogen-SK complex (BRL 26921) in patients with acute myocardial infarction (AMI). In the first study, 20 patients received either 750,000 units (U) SK (group IA, n = 10) or 1,500,000 U SK (group IB, n = 10) within 5–10 min intravenously. In the second study 10 consecutive patients received 750,000 U SK within 15 min (group IIA) intravenously. The following 10 consecutive patients received 30 mg BRL 26921 within 2 min (group IIB). Early reperfusion was found in 16 patients in the first study (8 in each group) and in 18 patients in the second study (9 in each group). The decrease of fibrinolytic activity was biphasic with a half-disappearance time of 112.5 min for BRL 26921 and 31 (IA) and 18 (IB) min for SK. α2-Antiplasmin depletion and a decrease of fibrinogen was observed with no differences after bolus injections of SK and of BRL 26921.
A Hereditary Dysfibrinogenemia: Fibrinogen Awaji
Authors: Matsuo T. , Okuno S. , Mukaida T. , Ueshima S. , Okada K. , Matsuo O.
Published: April 2009

Abnormal function of fibrinogen was observed in a 2 5-year-old woman with no symptoms attributable to dysfibrinogenemia. Disturbed polymerization of fibrin monomer was identified, but the release of fibrinopeptide from the purified fibrinogen and the cross-linking by factor XIII were normal. Other abnormal findings included a high value of fibrinogen degradation products, rapid mobility on immunoelectrophoresis and abnormal spot of γ-chain on two-dimensional polyacrylamide gel electrophoresis. Similar abnormalities were also observed among the patient’s family members.
Effect of Oral Anticoagulant Treatment on Plasma and Serum Antithrombin III: A Study on 172 Patients at Different Levels of Anticoagulation
Authors: De Stefano V. , Leone G. , De Martini D. , Di Donfrancesco A. , Accorrà F. , Bizzi B.
Published: April 2009

Antithrombin III (AT III) functional levels are much lower in serum than in plasma; during oral anticoagulation this difference is reduced. Plasma and serum of 172 patients taking vitamin K antagonists were tested for AT III antigen and both AT III heparin cofactor and anti-Xa heparin cofactor. Crossed immunoelectrophoresis of AT III on heparin-agarose was also carried out in plasma and serum. The patients were divided into four groups: (1) international normalized ratio (INR) 9.3–4.1, n = 25; (2) INR 4.0–2.5, n = 73; (3) INR 2.4–2.0, n = 40, and (4) INR 1.9–1.5, n = 34. 66 healthy subjects were used as controls. Plasma levels of AT III antigen, AT III heparin cofactor, and anti-Xa heparin cofactor were the same in all groups. In all groups all serum AT III parameters were higher than in controls; crossed immunoelectrophoresis of AT III on heparin-agarose indicated that this finding was due to a lower formation of complexed AT III in serum. AT III heparin cofactor serum values were the same whatever the INR over a large range (9.3–1.5); the highest anti-Xa heparin cofactor serum levels were noted in the groups treated more intensely (groups 1 and 2).
Analysis of Platelet von Willebrand Factor Antigen
Authors: Thomas Kathy B. , Firkin Barry G. , Howard Margaret A.
Published: April 2009

Platelet lysates were obtained from suspensions of normal washed platelets by freeze-thawing or Triton X-100 lysis. The resultant platelet lysates contained 0.34 ± 0.15 U/109 platelets (n = 8) of von Willebrand factor antigen (vWf:Ag) as determined by radio-electroimmunoassay using a monospecific antibody to vWf:Ag. The vWf:Ag level was higher in platelet lysates prepared from freshly drawn blood than from outdated platelet packs. Platelet lysates from patients with severe von Willebrand’s disease type I (n = 2) did not contain detectable vWf:Ag. When normal platelet lysates were analyzed by radiocrossed immunoelectrophoresis in agarose using a monospecific polyclonal antibody to plasma vWf:Ag, two immunochemically identical precipitin peaks were seen. One of the platelet vWf:Ag peaks corresponded in its electrophoretic mobility to plasma vWf:Ag, while the other peak, i.e. platelet vWf:Ag-peak II, migrated to a more anodal position. The presence of the platelet vWf:Ag-peak II suggests structural differences between plasma and platelet vWf:Ag and illustrates previously unrecognized heterogeneity of platelet vWf:Ag.
Release of Plasminogen Activator by Batroxobin
Authors: Klöcking H.-P. , Hoffmann Anna , Markwardt F.
Published: April 2009

In the isolated perfused pig ear, batroxobin caused a dose-dependent increase in the release of plasminogen activator. The activator-releasing effect required the presence of the active enzyme. The activator released was of the tissue-type.
Effects of Pro-Urokinase and Urokinase on the Fibrinolytic System in Man
Authors: Trübestein Gustav , Popov Smilja , Wolf Helmut , Welzel Dieter
Published: April 2009

The fibrinolytic and fibrinogenolytic properties of kidney cell pro-urokinase (PUK) were compared with those of natural urinary urokinase in human volunteers. Comparable degrees of fibrinolysis were obtained at a concentration of 500,000 U UK and a concentration of 500,000 IU natural urokinase. Natural urokinase showed a strong activation of the fibrinolytic system in plasma, evidenced by plasminogen activation, α2-antiplasmin consumption, and the rise in fibrinogen-fibrin degradation products. In contrast to UK, there was no fall in plasminogen, no consumption of α2-antiplasmin, and only a slight amount of fibrinogen-fibrin degradation products produced with PUK. These findings with PUK reveal a high affinity for fibrin and may result in better clot selectivity.
In vitro and ex vivo Effects of Indobufen on Human Platelet Aggregation, the Release Reaction and Thromboxane B2 Production
Authors: Cattaneo M. , Bevilacqua C. , Lecchi A. , Mannucci P.M.
Published: April 2009

We have done a comprehensive study in normal volunteers of the in vitro and ex vivo effects of the antiplatelet agent indobufen on platelet aggregation, the release reaction and thromboxane B2 (TxB2) production as induced by different concentrations of aggregating agents. At low concentrations (10 μM), indobufen completely inhibited secondary platelet aggregation, the release reaction and TxB2 production stimulated by ADP, epinephrine and low concentrations of platelet-activating factor (PAF acether). Higher concentrations of indobufen (100 μM) completely inhibited TxB2 production, platelet aggregation and ATP release induced by arachidonic acid (1 mM) or collagen (2 μg/ml). The inhibitory effect was partially overcome by higher concentrations of arachidonic acid (2 mM). Data obtained ex vivo 2 h after the oral administration of 200 mg indobufen to 8 normal volunteers were in keeping with those of the in vitro study. We conclude that indobufen inhibits platelet aggregation and the release reaction by inhibiting the platelet arachidonate pathway.
Elevated Fibrinopeptide A Levels in Patients with Clinically Localised Breast Carcinoma
Authors: Auger M.J. , Galloway M.J. , Leinster S.J. , McVerry B.A. , Mackie M.J.
Published: April 2009

58 patients with clinically localised breast carcinoma, treated by either mastectomy alone or lumpectomy plus local radiotherapy, have now been followed for up to 51 months (median 12 months). 21 of the 58 patients (36.2%) had a persistently elevated or rising fibrinopeptide A level after surgery and 11 of these patients (52%) to date have subsequently developed recurrent breast carcinoma up to 27 months after the fibrinopeptide A level became elevated (median 6 months). Conversely, only 2 (5.4%) of the 37 patients with persistently normal post-operative fibrinopeptide A levels have developed recurrent disease. Elevated fibrinopeptide A levels appear to be a marker of persistent tumour activity and precede clinical recurrence in certain patients with breast carcinoma, but normal values do not exclude recurrent or residual disease.
In vitro Effect of a Microfibrillar Collagen Hemostat on Platelets
Authors: McClure M. , Duncan G.D. , Born G.V.R. , Robicsek F.
Published: April 2009

Microscopic particles of microfibrillar collagen hemostat (MCH) have been shown to pass through a 40-μm blood transfusion filter. This study indicates that MCH passing through the filter retains a significant portion of its ability to aggregate platelets in vitro and the aggregating ability is dose related. The extent of platelet aggregation in the presence of the MCH filtrate or an ADP standard was measured photometrically and log dose-response curves constructed from the data. An ED50 of 3.6 μg protein/ml platelet-rich plasma (PRP) was calculated at a protein concentration of 7.6 mg/ml in the MCH filtrate and an ED50 of 5.9 μg protein/ml PRP at 12 mg/ml protein in the MCH filtrate. The platelet aggregating ability of the MCH filtrates corresponded to 18.8 and 39.6% of the maximum response produced by adenosine-5’-diphosphate. These findings support the view that blood contaminated with MCH should not be returned to a patient’s circulation.
Monocyte Procoagulant Activity as a Peripheral Marker of Clotting Activation in Cancer Patients
Authors: Morgan D. , Edwards R.L. , Rickles F.R.
Published: April 2009

Peripheral blood monocytes generate the procoagulant tissue factor in vitro and in vivo in response to stimulation by a variety of agents. Monocytes from cancer patients generate significantly increased tissue factor and a quantitative relationship exists between the levels of monocyte tissue factor (MTF) and levels of circulating fibrinopeptide A (FPA), a marker of in vivo clotting activation. Furthermore, monocytes from cancer patients have a greater procoagulant response to stimulation by endotoxin in vitro, which appears independent of lymphocyte regulation. These findings suggest a priming process in vivo, and may reflect exposure of monocytes to tumor antigen(s) or components of the immune response to tumors.
Effects of Nicotine on Platelet Function
Authors: Pfueller Sharron L. , Burns Paul , Mak Kevin , Firkin Barry G.
Published: April 2009

In non-smokers, only high concentrations of nicotine (10 rs\M) caused platelet aggregation in platelet-rich plasma and release of 5-hydroxytryptamine (5-HT). Both responses to ADP and 5-HT were enhanced at 1 and 10 mM nicotine while they were inhibited to collagen, ristocetin, adrenaline and arachidonic acid. Lower concentrations of nicotine had no effect with any agent other than 5-HT, with which a variable enhancement of 5-HT-induced aggregation was observed. Uptake of 14C-5-HT was inhibited by nicotine at 100 μM or higher while platelet factor 3 availability was unaffected. Thus it is unlikely that direct effects of nicotine on platelets are responsible for smoking-related changes in platelet reactivity.
Intravascular Coagulation Phenomena Associated with Prevalent Fall in Fibrinogen and Plasminogen during L-Asparaginase Treatment in Leukemic Children
Authors: Legnani C. , Palareti G. , Pession A. , Poggi M. , Vecchi V. , Bianchini B. , Coccheri S.
Published: April 2009

12 children affected by acute lymphoblastic leukemia had higher baseline levels of Normotest, Antithrombin III activity (AT III:A) and antigen (AT IIl·Ag) than those found in the control group, due to increased liver protein synthesis, as shown by the higher values of 30511bumin. Treatment with L-asparaginase (L-Asp) induced a marked decrease in fibrinogen and plasminogen and only a slight but significant reduction in AT III:A, AT III:Ag, protein C and α2 antiplasmin levels. Platelet counts progressively increased. Each single L-Asp administration acutely induced (as recorded in the 2-hour postadministration samples) a significant increase in fibrinopeptide A values and reduction in several factors and inhibitors, due to activation of coagulation and consumption phenomena. It is suggested that these acute changes, but not the marked and selective reduction in fibrinogen and plasminogen, were partly compensated during L-Asp treatment by an overall increase in clotting and nonclotting protein synthesis in the liver.
Control of Human Plasminogen Activation
Authors: Castellino F.J. , Urano T. , de Serrano V. , Morris J.P. , Chibber B.A.K.
Published: April 2009

The activation of GlU1-plasminogen (Glu-Pg) by streptokinase (SK), urokinase (UK) and tissue plasminogen activator (tPA) is under rigorous control by molecules such as ε-aminocaproic acid (EACA), fibrinogen (Fg), fibrin (Fn) and, as we have recently discovered, anions. This presentation will focus on the biochemical mechanisms that are involved in these processes. In the case of activation by SK, a species of activator complex, composed of Glu-Pg and SK, can be identified that is inhibited by anions, such as C1–, and stimulated by Fg and Fn. This species rapidly decays to another activator complex, also consisting of Glu-Pg and SK, that is much less sensitive to control by these effector molecules. The most stable activator complex, containing equimolar SK and plasmin, is not affected to a great extent by anions, Fg or Fn. In the overall activation of Glu-Pg by SK, C1– behaves as a mixed inhibitor, with a Ki of 6.4–9.2 mM, and Fg functions as a mixed activator, displaying a Ka of 110–240 nM. These results show that activation of Glu-Pg by SK in physiological samples would be considerably inhibited by C1– in the absence of Fg. The activation of Glu-Pg by both high- and low-molecular weight UK is also inhibited by C1–, but is stimulated by EACA. The inhibition by C1– does not occur in the presence of concentrations of EACA that saturate its weak binding sites on Glu-Pg, and the stimulation by EACA is maximally exhibited in the presence of C1–. The anion inhibitory effect follows a Hofmeister series, as does the binding (stimulatory) constant of EACA to Glu-Pg. Based upon structural studies of Glu-Pg in the presence of a variety of anions, we find that Glu-Pg can adopt a conformation that is optimal to activation and one that is not. The former occurs in the presence of weakly bound anions, or in the presence of any salt, plus saturating levels of EACA. The latter conformation is adopted in the presence of tightly bound anions. Anions also influence the rate of Glu-Pg activation by tPA, and the stimulation of this process by Fg and Fn. The mechanism of this effect is currently under investigation.
Effect of Anti Plasminogen Monoclonal Antibodies on Whole Blood Clot Lysis
Authors: Ehrenreich F. , Hattey E. , Wojta J. , Binder B.R.
Published: April 2009

Several monoclonal antibodies (MABs) against specific parts of the plasminogen molecule were developed. One of them, MPWIPG, recognizes only the native form Glu-plasminogen, while the other one, MPW2PG, reacts equally well with Glu-plasminogen and its proteolytically degraded derivative Lys-plasminogen as confirmed by immunoblotting experiments. Both MABs do not alter the cleavage of the low molecular weight substrate S-2251 by plasmin in a purified system but rather increase the rate of plasmin formation by urokinase and t-PA in a system without fibrin. In the presence of fibrin MPWIPG has no effect on plasmin formation by t-PA, while MPW2PG exhibits mixed type inhibition. To investigate whether this effect can also be seen in a whole blood clot lysis system, the Chandler loop was used (PVC tubes, 4 mm inner diameter, 28 mm length). 125I-fibrinogen was added to citrated whole blood (0.36 % sodium citrate) to about 5000 cpm/10 ul sample. 1.5 ml of the mixture were pipetted into each tube, the tubes were closed, put on a rotary plate, tilted to an angle of 23°, and rotated at 16 rpm. 10 ul samples were taken for radioactive counting before clotting by addition of 10 ul 3.02M CaCl2 (100 %-value), 45 min after clotting (0 %-value), and 30, 60, 120, 180, 360 minutes after addition of activators. Lysis was started one hour after recalcification. Different amounts of MABs were added either 30 min before recalcification (MAB endogen) or together with the activator (MAB exogen) Percent lysis was calculated from the radioactivity released into the fluid phase. Double determinations were done in all experiments. Streptokinase (50 IU/ml final), u-PA (100 or 500 IU/ml final) and t-PA (100 ng/ml final) were used as plasminogen activators. Streptokinase-induced lysis was stimulated by the antibodies (70 ug/ml) with a stimulation index of 2.7 for MPWIPG and 1.7 for MPW2PG, respectively. Urokinase induced lysis was also stimulated by both antibodies but in a dose-dependent fashion and about 6-fold. t-PA mediated lysis was stimulated by both MABs but not in a dose dependent way.
Review of Studies with Plasminogen Concentrates and Proposals for Further Therapeutic Strategies with Plasminogen Concentrates
Authors: Anderle K. , Fröhlich A.
Published: April 2009

Since the introduction of thrombolytic treatment based on the activation of plasminogen (PLG) by streptokinase (SK) and urokinase (UK) the search for new and improved methods has been continuing. The pivotal issue is how to achieve clot-specific fibrinolysis without producing systemic fibrinogenolysis. One out of various approaches to enhance lysis rates has been the use of PLG either alone or in combination with UK or SK in the light of the fact that fibrinolytic treatment, particularly using SK, is accosiated with a consumption of PLG, and that thrombi contain relatively small amounts of native PLG, however, are capable of incorporating added PLG in vitro. PLG-concentrates from various manufactures have been administered intravenously for treatment of deep venous thrombosis, mainly in combination with SK, and of pulmonary embolism in combination with UK. Local intracoronary and intraarterial administration in combination with UK has been reported in patients with myocardial infarction, and peripheral arterial occlusions, respectively. Lysis rates obtained in these studies were in most cases superior to results obtained with SK or UK alone, without increasing the incidence of bleeding complications. In addition, excellent results in a larger group of patients with cerebral thrombosis were obtained with PLG alone. The encouraging results of these studies may be explained by the fact that all of the preparations used contained partially activated forms of PLG (commonly designated lys-PLG) to a greater or lesser extent. Lys-PLG has a higher affinity for fibrin than the native glu-PLG and is activated by UK or SK by a manyfold faster. These properties allow for a rapid formation of plasmin which – bound to fibrin – is also protected from the attack of neutralizing antiplasmin. The design and results of previous studies with lys-PLG concentrates will be reviewed and approaches to further improve fibrinolytic regimens with lys-PLG-concentrates discussed.
Evaluation of the Biological Effects of Low-Molecular-Weight Heparins
Authors: Bender N. , Seidl C. , Weichert W. , Breddin HK.
Published: April 2009

In experiments with a laser thrombosis model in rat mesenteric venules, where laser beams induce defined injuries at the vessel endothelium, antithrombotic potency of low-molecular-weight (LMW) heparins was calculated by the number of laser injuries required to induce vessel occluding thrombi. The antithrombotic effect of various LMW heparins was measured at defined time intervals after subcutaneous application. Simultaneously, rat blood was collected and aXa and aIIa plasma levels were determined by chromogenic substrate assays. The antithrombotic effect calculated in the animal model lasted longer than 48 h after a single subcutaneous heparin injection whereas the anticoagulative effect measured as aXa or aIIa activity was measurable only within the first 6 h after subcutaneous application. LMW heparins showed an antithrombotic effect at dosages where no influence on the coagulation system could be demonstrated.
Experimental Investigations on the Thrombosis-Preventing Effect of Low-Molecular-Weight Heparins
Authors: Harbauer G. , Allendorf A.
Published: April 2009

6 different preparations of low-molecular-weight heparin (LMWH) derivatives and 1 unfractioned heparin (UFH) were tested for their thromboprophylactic effects in an experimental model which closely simulates clinical conditions. Each preparation was tested in 5 rabbits 2 h and in 5 rabbits 12 h after subcutaneous application of 20–200 U/kg body weight (according to the suggestions of the manufacturers). The animals were anesthetized with intravenous pentobarbital sodium about 90 min or 11.5 h after the application of the heparin preparation. The right external jugular vein was dissected free and a thrombogenesis was provoked. There was no significant protection against occluding thrombosis 2 h after LMWH or UFH injection, when plasma levels of heparin-like activities were found to be high in another experimental series. Surprisingly, there were definite thromboprophylactic effects 12 h (when heparin-like plasma activities had disappeared) after application of 4 (out of 6) LMWH preparations. No protection against thrombosis was seen 12 h after application of UFH.
Dependence of the So-Called Heparin Availability on Experimental Factors
Authors: Stemberger A. , Haas S. , Blümel G.
Published: April 2009

The so-called heparin level in the plasma of patients or healthy volunteers should be determined using only those methods which correspond to the test standard used by the producer of the heparin applied or which approach the corresponding test principle. The correlation of the ex vivo findings gained through biochemical and functional methods to the physiological effect is, as far as possible, only then guaranteed when methodical influences can be excluded. The data obtained through these test series do not, however, allow a transference to the physiological effect of the corresponding heparin preparations regarding anticoagulant as well as antithrombotic efficacy.
Prophylaxis of Thromboembolism with Various Low-Molecular-Weight Heparins
Authors: Haas S. , Blümel G.
Published: April 2009

In numerous clinical studies, the antithrombotic effect and tolerance of low-molecular-weight heparins (LMWHs) were compared to those of unfractionated heparin (UFH). Based on our own experience and on a review of the literature the following conclusions can be drawn. In the field of prophylaxis of thromboembolism, including high-risk patients with major bone surgery, the antithrombotic efficacy of a single daily dose of LMWH is at least equivalent to two or three injections of UFH per day. It should, however, be noted that the margin between thromboembolic and bleeding complications has not become bigger, although this had been derived from experimental results. In order to avoid bleeding complications, it is absolutely necessary to take note of the manufacturers’ recommendations. This is particularly important when a change of various preparations is considered, because the standardization of LMWH preparations is different and the units are not comparable. The main advantage of LMWH results from an improved practicability. The single daily dose leads to an improved compliance of patients and the nursing staff is spared a great deal of work.
The Calcium/Calmodulin/Kinase System and Arrhythmogenic Afterdepolarizations in Bradycardia-Related Acquired Long-QT Syndrome
Authors: Qi XiaoYan , Yeh Yung-Hsin , Chartier Denis , Xiao Ling , Tsuji Yukiomi , Brundel Bianca J.J.M. , Kodama Itsuo , Nattel Stanley
Published: April 2009

Background—Sustained bradycardia is associated with long-QT syndrome in human beings and causes spontaneous torsades de pointes in rabbits with chronic atrioventricular block (CAVB), at least partly by downregulating delayed-rectifier K+-current to cause action potential (AP) prolongation. We addressed the importance of altered Ca2+handling, studying underlying mechanisms and consequences.

Methods and Results—We measured ventricular cardiomyocyte [Ca2+]i(Indo1-AM), L-type Ca2+-current (ICaL) and APs (whole-cell perforated-patch), and Ca2+-handling protein expression (immunoblot). CAVB increased AP duration, cell shortening, systolic [Ca2+]itransients, and caffeine-induced [Ca2+]irelease, and CAVB cells showed spontaneous early afterdepolarizations (EADs).ICaLdensity was unaffected by CAVB, but inactivation was shifted to more positive voltages, increasing the activation-inactivation overlap zone forICaLwindow current. Ca2+-calmodulin–dependent protein kinase-II (CaMKII) autophosphorylation was enhanced in CAVB, indicating CaMKII activation. CAVB also enhanced CaMKII-dependent phospholamban-phosphorylation and accelerated [Ca2+]i-transient decay, consistent with phosphorylation-induced reductions in phospholamban inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase as a contributor to enhanced SR Ca2+loading. The CaMKII-inhibitor KN93 reversed CAVB-induced changes in caffeine-releasable [Ca2+]iandICaLinactivation voltage and suppressed CAVB-induced EADs. Similarly, the calmodulin inhibitor W7 suppressed CAVB-inducedICaLinactivation voltage shifts and EADs, and a specific CaMKII inhibitory peptide preventedICaLinactivation voltage shifts. The SR Ca2+-uptake inhibitor thapsigargin and the SR Ca2+release inhibitor ryanodine also suppressed CAVB-induced EADs, consistent with an important role for SR Ca2+loading and release in arrhythmogenesis. AP-duration changes reached a maximum after 1 week of bradypacing, but peak alterations in CaMKII and [Ca2+]irequired 2 weeks, paralleling the EAD time course.

Conclusions—CAVB-induced remodeling enhances [Ca2+]iload and activates the Ca2+-calmodulin-CaMKII system, producing [Ca2+]i-handling abnormalities that contribute importantly to CAVB-induced arrhythmogenic afterdepolarizations.