Characterization of the Rabbit as an In Vitro and In Vivo Model to Assess the Effects of Fibrinogenolytic Activity of Snake Venom on Coagulation.

Several in vitro investigations have demonstrated that anticoagulant effects of fibrinogenolytic snake venom metalloproteinases have been abrogated in human plasma by modifying fibrinogen with iron (Fe) and carbon monoxide (CO) to prevent catalysis or by directly inhibiting these enzymes with CO. To translate these findings, we chose to assess the rabbit as a model of envenomation with Crotalus atrox venom. It was determined with thrombelastography that 15 times the concentration of venom noted to compromise coagulation in plasma in vitro was required to cause coagulopathy in vivo, likely secondary to venom binding to blood cells and being cleared from the circulation rapidly. Unlike human plasma, rabbit plasma pre-treated with Fe/CO was not protected from fibrinogenolysis by venom. Consequently, the administration of purified human fibrinogen (with or without Fe/CO) would be required before venom administration to rabbits. Of greater interest, venom exposed to CO had complete loss of fibrinogenolytic effect in rabbit plasma and partial loss of activity in whole blood, indicative of unbinding of CO from venom and binding to haemoglobin. Thus, venom exposed to CO could remain partially or completely inhibited in whole blood long enough for clearance from the circulation, allowing rabbits to be a useful model to test the efficacy of regional CO administration to the bite site. Future investigations are planned to test these novel approaches to attenuate venom-mediated coagulopathy in the rabbit.

Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms.

: Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 µg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).

Effect of iron and carbon monoxide on fibrinogenase-like degradation of plasmatic coagulation by venoms of four Crotalus species.

Annually, thousands suffer poisonous snake bite, often from defibrinogenating species. Iron and carbon monoxide (CO) improve coagulation kinetics by modulation of fibrinogen as demonstrated in various Agkistrodon species and Crotalus atrox. Thus, we sought to determine whether pretreatment of plasma with iron and CO could attenuate venom-mediated catalysis of fibrinogen obtained from four common Crotalus species with known fibrinogenase activity. Human plasma was pretreated with ferric chloride (0-10 µmol/l) and CO-releasing molecule-2 (0-100 µmol/l) prior to exposure to venom from a Northern Pacific rattlesnake, Arizona black rattlesnake, prairie rattlesnake, or red diamond rattlesnake. The concentration of venom used decreased coagulation function of one or more kinetic parameters by at least 50% of normal values. Coagulation kinetics were determined with thrombelastography.Three snake venoms significantly degraded plasmatic coagulation kinetics, prolonging the onset to clot formation, diminishing velocity of clot growth and decreasing clot strength. However, red diamond rattlesnake venom exposure resulted in mixed coagulation kinetics, significantly decreasing the time to onset of coagulation without decreasing the velocity of clot growth. Iron and CO attenuated these coagulation kinetic changes in a species-specific manner. Further in vitro investigation of other fibrinogenolytic venoms is indicated to determine if iron and CO can attenuate venom compromised coagulation.

Effect of Iron and Carbon Monoxide on Fibrinogenase-like Degradation of Plasmatic Coagulation by Venoms of Six Agkistrodon Species.

Annually, thousands suffer poisonous snakebite, often from defibrinogenating species. It has been demonstrated that iron and carbon monoxide change the ultrastructure of plasma thrombi and improve coagulation kinetics. Thus, this investigation sought to determine whether pre-treatment of plasma with iron and carbon monoxide could attenuate venom-mediated catalysis of fibrinogen obtained from Agkistrodon species with fibrinogenase activity. Human plasma was pre-treated with ferric chloride (0-10 µM) and carbon monoxide-releasing molecule-2 (CORM-2, 0-100 µM) prior to exposure to 0.5-11 µg/ml of six different Agkistrodon species' venom. The amount of venom used for experimentation needed to decrease coagulation function of one or more kinetic parameters by at least 50% of normal values for (e.g. half the normal speed of clot formation). Coagulation kinetics were determined with thrombelastography. All six snake venoms degraded plasmatic coagulation kinetics to a significant extent, especially prolonging the onset to clot formation and diminishing the speed of clot growth. Pre-treatment of plasma with iron and carbon monoxide attenuated these venom-mediated coagulation kinetic changes in a species-specific manner, with some venom effects markedly abrogated while others were only mildly decreased. Further in vitro investigation of other pit viper venoms that possess fibrinogenolytic activity is indicated to identify species amenable to or resistant to iron and carbon monoxide-mediated attenuation of venom-mediated catalysis of fibrinogen. Lastly, future pre-clinical investigation with animal models (e.g. rabbit ear-bleed model) is planned to determine whether iron and carbon monoxide can be used therapeutically after envenomation.

Iron and carbon monoxide attenuate Crotalus atrox venom-enhanced tissue-type plasminogen activator-initiated fibrinolysis.

In addition to degrading fibrinogen as a source of consumptive coagulopathy, rattlesnake venom has also been demonstrated to enhance fibrinolysis and degrade alpha-2-antiplasmin. The goals of this investigation was to characterize the kinetic fibrinolytic profile of Crotalus atrox venom in the absence and presence of tissue-type plasminogen activator (tPA), and to also ascertain if iron and carbon monoxide (CO, a positive modulator of alpha-2-antiplasmin) could attenuate venom-enhanced fibrinolysis. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to C. atrox venom (0-2 µg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, either separately or in combination, plasma was exposed to iron (ferric chloride, 10 µmol/l) or CO (carbon monoxide-releasing molecule-2, 100 µmol/l) prior to incubation with venom; the plasma sample was subsequently subjected to thrombelastographic analysis with addition of tPA. Venom exposure in the absence of tPA did not result in detectable fibrinolysis. In the presence of tPA, venom markedly enhanced fibrinolysis. Iron and CO, markedly attenuated venom enhancement of fibrinolysis. C. atrox venom enhances tPA-mediated fibrinolysis, and interventions that enhance/protect alpha-2-antiplasmin activity significantly attenuate venom-enhanced fibrinolysis. Future preclinical investigation is required to determine if iron and CO can attenuate venom-mediated degradation of alpha-2-antiplasmin-dependent fibrinolytic resistance.

Effects of profound hypoxemia on coagulation & fibrinolysis in normal individuals.

Hypoxia has been proposed to enhance, diminish, or have no effect on laboratory measures of coagulation or clinical thrombosis. Further, there usually are significant pathological or environmental factors concurrently present with hypoxia. Thus, the goal of the present investigation was to determine whether whole blood or plasmatic coagulation and fibrinolytic kinetics would change in response to progressive hypoxia to a systemic oxygenation (SpO2) of 70%. Healthy, conscious volunteers (n = 9) breathing a hypoxic mixture of gases during an in-vivo validation of noninvasive cerebral oximetry had blood samples collected and assessed with thrombelastography at normoxia and after SpO2 of 70%. A mild release of endogenous heparin-like activity occurred that diminished plasmatic coagulation, and a mild increase in clot lysis time also was noted. Further investigation to determine whether these phenomena occur in more chronic, less hypoxic states as sources of hypocoagulation or thrombophilia is needed.

Left atrial myxoma presenting as pulmonary embolism: potential role of heme oxygenase-1.

We present the case of a patient with left atrial myxoma that presented with pulmonary embolism. The patient did not have any intracardiac communication between right and left sides of the heart. Using thrombelastography, the patient was determined to have an abnormally large velocity of plasma thrombus growth and strength with reduced vulnerability to lysis. Critically, increased carboxyhemoglobin concentrations were present, likely secondary to hemolysis from the tumor and engagement of systemic heme oxygenase-1. It was determined that the patient's plasmatic hypercoagulability was in part due to carboxyhemefibrinogen formation via a thrombelastographic method. In addition to circulating hypercoagulability, the patient also had an area of chronic venous stasis in his left ankle that had not changed for over a decade prior to this thrombophilic episode. In conclusion, we present the first case of paradoxical pulmonary embolism in the presence of a left atrial myxoma, potentially secondary to a combination of hemolysis, heme oxygenase-1 up-regulation, systemic hypercoagulability/hypofibrinolysis, and regional venous stasis.

Accuracy of acoustic respiration rate monitoring in pediatric patients.

BACKGROUND: Rainbow acoustic monitoring (RRa) utilizes acoustic technology to continuously and noninvasively determine respiratory rate from an adhesive sensor located on the neck. OBJECTIVE: We sought to validate the accuracy of RRa, by comparing it to capnography, impedance pneumography, and to a reference method of counting breaths in postsurgical children. METHODS: Continuous respiration rate data were recorded from RRa and capnography. In a subset of patients, intermittent respiration rate from thoracic impedance pneumography was also recorded. The reference method, counted respiratory rate by the retrospective analysis of the RRa, and capnographic waveforms while listening to recorded breath sounds were used to compare respiration rate of both capnography and RRa. Bias, precision, and limits of agreement of RRa compared with capnography and RRa and capnography compared with the reference method were calculated. Tolerance and reliability to the acoustic sensor and nasal cannula were also assessed. RESULTS: Thirty-nine of 40 patients (97.5%) demonstrated good tolerance of the acoustic sensor, whereas 25 of 40 patients (62.5%) demonstrated good tolerance of the nasal cannula. Intermittent thoracic impedance produced erroneous respiratory rates (>50 b·min(-1) from the other methods) on 47% of occasions. The bias ± SD and limits of agreement were -0.30 ± 3.5 b·min(-1) and -7.3 to 6.6 b·min(-1) for RRa compared with capnography; -0.1 ± 2.5 b·min(-1) and -5.0 to 5.0 b·min(-1) for RRa compared with the reference method; and 0.2 ± 3.4 b·min(-1) and -6.8 to 6.7 b·min(-1) for capnography compared with the reference method. CONCLUSIONS: When compared to nasal capnography, RRa showed good agreement and similar accuracy and precision but was better tolerated in postsurgical pediatric patients.

Transfusion protocol for implantation and explantation of cardiac devices in an academic medical center.

Few reports in the literature describe the transfusion support needed for implantation and explantation of cardiac assist devices. Because such devices are frequently used at this institution, we have established a protocol to support these patients. We reviewed blood bank records to document the usage of blood components in all patients on cardiac assist devices from 1989 to 2005 (n = 215). The data were separated into components given at the time of implantation and explantation of the device. Implantation data on 214 patients showed the mean use as follows: red blood cells (RBC), 5.7 U; platelets (PLT), 2.4 U; fresh frozen plasma (FFP), 7.5 U; and cryoprecipitate, 0 U. Explantation data on 134 patients showed a mean use as follows: RBC, 7.7 U; PLT, 2.4 U; FFP, 9.9 U; and cryoprecipitate, 3.7 U. Our protocol for the preparation of blood components for implantation and explantation of a cardiac device is, implantation: RBC, 4 U; PLT, 1 U; FFP, 6 U; explantation: RBC, 8 U; PLT, 2 U; FFP, 8 U. Our data do not support the need for preparation of cryoprecipitate before implantation or explantation.