Vance G. Nielsen

Effects of coagulation factor deficiency on plasma coagulation kinetics determined via thrombelastography®: critical roles of fibrinogen and factors II, VII, X and XII

Background:  Thrombelastography (TEG®) is used to assess coagulopathy. However, a comprehensive characterization of the effects of specific coagulation factor deficiencies and mode of activation on TEG® data does not exist.

Colloids decrease clot propagation and strength : role of factor XIII-fibrin polymer and thrombin-fibrinogen interactions

Colloid‐mediated hypocoagulability is clinically important, but the mechanisms responsible for coagulopathy have been incompletely defined. Thus, my goal was to elucidate how colloids decrease plasma coagulation function. Plasma was diluted 0% or 40% with 0.9% NaCl, three different hydroxyethyl starches (HES, mean molecular weight 450, 220 or 130 kDa), or 5% human albumin. Samples (n = 6 per condition) were activated with celite, and diluted samples had either no additions or addition of fibrinogen (FI), thrombin (FIIa) or activated Factor XIII (FXIIIa) to restore protein function to prediluted values. Thrombelastographic variables measured included clot propagation (angle, α), and clot strength (amplitude, A; or shear elastic modulus, G). Dilution with 0.9% NaCl significantly decreased α, A and G‐values compared to undiluted samples. Supplementation with FI, but not FIIa or FXIIIa, resulted in 0.9% NaCl‐diluted thrombelastographic variable values not different from those of undiluted samples. FI supplementation of HES 450, HES 220, HES 130 and albumin‐diluted samples only partially restored α, A and G‐values compared to undiluted samples. FIIa addition only improved clot propagation and strength in albumin‐diluted samples. FXIIIa supplementation improved propagation in samples diluted with HES 450, HES 220 and albumin, and clot strength improved in HES 450 and albumin‐diluted plasma. Considered as a whole, these data support compromise of FIIa‐FI and FXIIIa – fibrin polymer interactions as the mechanisms by which colloids compromise plasma coagulation. Investigation to determine if clinical enhancement of FXIII activity and/or FI concentration (e.g. fresh‐frozen plasma, cryoprecipitate) can attenuate colloid‐mediated decreases in hemostasis is warranted.

A comparison of the Thrombelastograph and the ROTEM

Elastic modulus-based assessment of hemostasis in clinical and research settings has been conducted for nearly 60 years. Two systems utilizing this technology include the Thrombelastograph (Haemoscope Corporation, Niles, Illinois, USA) and the ROTEM (Pentapharm GmbH, Munich, Germany). The study goal was to compare the Thrombelastograph and the ROTEM to determine whether differences in data could be detected. Plasma (n = 8 per condition) was not activated (native); was celite-activated, hypocoagulable by dilution with hydroxyethyl starch; was celite-activated, normal; and was celite-activated, hypercoagulable by addition of fibrinogen. Equivalent ratios of plasma mixture to CaCl2 were maintained between the Thrombelastograph and ROTEM systems. Data collected included the reaction time, angle, maximum amplitude and maximum elastic modulus. The ROTEM system generated significantly (P < 0.05) smaller reaction time (mean ± SD, 459 ± 39 versus 788 ± 94 s) and greater angle (60.1 ± 5.3 versus 46.4 ± 4.6°), maximum amplitude (27.6 ± 1.6 versus 24.4 ± 1.4 mm) and maximum elastic modulus (1911 ± 152 versus 1616 ± 121 dynes/cm2) values with native plasma compared with the Thrombelastograph. Celite-activation tended to attenuate differences between the two systems. In conclusion, a comparison of the Thrombelastograph and the ROTEM with similar samples demonstrated clinically significant differences in the data generated without exogenous activation. Given these data and the differences in proprietary activator, divergent results are expected, perhaps affecting clinical decision-making.

Evaluation of the contribution of platelets to clot strength by thromboelastography in rabbits: the role of tissue factor and cytochalasin D.

The contribution of platelets and soluble clotting components to clot strength has been the focus of several clinical studies using thromboelastography; it would, therefore, be beneficial to develop an animal model with which to mechanistically approach hemostatic disorders. Thus, we proposed to determine if the contribution of platelet function (GP, dyne/cm2) and soluble components of the coagulation pathway to total clot strength (GT) in rabbits were similar to those in humans. Blood was sampled from the ear arteries of conscious rabbits (n = 12); 350 &mgr;L of the blood was placed in a thromboelastograph. Ten microliters of normal saline, cytochalasin D (an inhibitor of microtubule function, 10 &mgr;M final concentration), or tissue factor (a potent stimulator of platelet function, 0.00625% final concentration) was added to the blood sample, and thromboelastography performed for 1 h. The GT (mean ± sd) was significantly (P < 0.001) different among samples exposed to normal saline, cytochalasin D, or tissue factor, with GT values of 7238 ± 1432, 937 ± 372, and 16,556 ± 3314, respectively. GP was responsible for 87% and 94% of GT in the absence or presence of tissue factor, respectively. GP did not significantly correlate with platelet concentration in the absence or presence of tissue factor. The contribution of GP to GT is similar to that observed in humans. Implications Rabbits may serve as a model of hemostasis that closely approximates human situations to mechanistically determine the etiology of coagulopathy. The contribution of platelet function to total clot strength is similar to that observed in humans.

Increased Injury Following Intermittent Fetal Hypoxia-Reoxygenation Is Associated with Increased Free Radical Production in Fetal Rabbit Brain

Hypoxia associated with perinatal events can result in brain damage in the neonate. In labor and eclampsia, hypoxia can be intermittent, which may result in more severe damage than sustained hypoxia. The pathogenesis of brain injury in sustained ischemia involves free radical production; therefore, we investigated whether higher levels of free radicals contribute to the greater injury induced by repetitive ischemia. Brains were obtained from fetuses of near-term, pregnant rabbits subjected to repetitive ischemia-reperfusion (RIR), sustained uterine ischemia-reperfusion (IR), or a control protocol. Compared with controls, fetal brains from RIR or IR groups had more brain edema. Brains from RIR fetuses exhibited higher levels of lipid peroxidation, 3-nitrotyrosine, and nitrogen oxides, and lower total antioxidant capacity and cortical cellular viability than those of IR or control fetuses. Maternal administration of antioxidants following RIR and fetal bradycardia resulted in lower levels of fetal cortical and hippocampal cell death. Coadministration of Trolox and ascorbic acid resulted in less brain edema and liquefaction, and fewer hippocampal ischemic nuclei as compared with the saline control. Higher free radical production may be responsible for the greater fetal brain injury following repetitive hypoxia-reoxygenation. Maternal antioxidant treatment resulted in transplacental passage of antioxidants and amelioration of brain injury, and may be a viable clinical option following diagnosis of fetal distress.

Elastic modulus-based thrombelastographic quantification of plasma clot fibrinolysis with progressive plasminogen activation

Thrombelastographic detection of fibrinolysis has been critical in the identification and treatment of coagulopathy in many perioperative settings. However, the fibrinolytic assessments have been at best non-parametric, amplitude-based determinations (e.g. estimated % lysis, clot lysis time or clot lysis rate). Recognizing this limitation, a methodology was developed to measure the onset, speed and extent of clot disintegration by changes in elastic modulus derived from the amplitude. Using this approach, our goal was to characterize the clot disintegration kinetics of progressive plasminogen activation with tissue plasminogen activator (tPA) and to determine the extent of inhibition of fibrinolysis mediated by tPA with aprotinin and activated factor XIII. While the estimated % lysis and clot lysis time were significantly affected by tPA (0–300 U/ml), elastic modulus-based analyses in a more activity-specific fashion demonstrated significantly decreased onset, increased rate and increased extent of fibrinolysis. Furthermore, aprotinin was found to inhibit the onset, rate and extent of fibrinolysis in an activity-dependent fashion, whereas activated factor XIII was noted to enhance the speed of onset of clot growth and delay the onset of fibrinolysis. In summary, our results serve as the rational basis to utilize this elastic modulus-based approach to quantify the extent of fibrinolysis in clinical and laboratory settings, as well as potentially guiding antifibrinolytic therapy.

cAMP activation of chloride and fluid secretion across the rabbit alveolar epithelium

Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl- movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl- transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl--free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl- and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl- secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+ absorptive pathways are damaged, transepithelial Cl- secretion and the consequent intra-alveolar fluid influx may be upregulated.Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl- movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl- transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl--free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl- and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl-secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+absorptive pathways are damaged, transepithelial Cl- secretion and the consequent intra-alveolar fluid influx may be upregulated.

Hextend (hetastarch solution) decreases multiple organ injury and xanthine oxidase release after hepatoenteric ischemia-reperfusion in rabbits.

OBJECTIVE We hypothesized that multiple organ injury and concentrations of xanthine oxidase (an oxidant-generating enzyme released after hepatoenteric ischemia) would be decreased by the administration of a bolus of a colloid solution at reperfusion. DESIGN Randomized, masked, controlled animal study. SETTING University-based animal research facility. SUBJECTS Fifty-four New Zealand white male rabbits, weighing 2 to 3 kg. INTERVENTIONS Anesthetized rabbits were assigned to either the hepatoenteric ischemia-reperfusion group (n = 27) or the sham-operated group (n = 27). Hepatoenteric ischemia was maintained for 40 mins with a balloon catheter in the thoracic aorta, followed by 3 hrs of reperfusion. Each group was randomly administered a bolus of one of three fluids at the beginning of reperfusion: Hextend (hetastarch solution); 5% human albumin; or lactated Ringers solution. The investigators were masked as to the identity of the fluid administered. MEASUREMENTS AND MAIN RESULTS Multiple organ injury was assessed by the release of lactate dehydrogenase activity into the plasma and by indices of gastric and pulmonary injury. Circulating lactate dehydrogenase activity was significantly greater (p < .001) in animals receiving lactated Ringers solution than in rabbits receiving either colloid solution. Gastric injury (tissue edema, Histologic injury Score) was significantly decreased (p < .01) by administration of both colloid solutions. Lung injury (bronchoalveolar lavage lactate dehydrogenase activity) was significantly decreased (p < .05) by the hetastarch solution administration. The hetastarch solution administration resulted in 50% less xanthine oxidase activity release during reperfusion compared with albumin or lactated Ringers solution administration (p < .001). CONCLUSION We conclude that multiple organ injury and xanthine oxidase release after hepatoenteric ischemia-reperfusion are decreased by colloid administration.

Gastric intramucosal pH and multiple organ injury: impact of ischemia-reperfusion and xanthine oxidase.

OBJECTIVES To determine if gastric intramucosal pH is affected by hepatoneteric ischemia-reperfusion. We additionally proposed to determine if changes in gastric mucosal hydrogen ion concentration are associated with liver and lung injury following hepatoenteric ischemia-reperfusion. Finally, we hypothesized that gastric intramucosal pH is influenced by xanthine oxidase, an oxidant-generating enzyme released after hepatoenteric ischemia-reperfusion. DESIGN Randomized, controlled, animal study. SETTING University-based animal research facility. SUBJECTS Thirty-six New Zealand white male rabbits (2 to 3 kg). INTERVENTIONS Anesthetized rabbits were randomly assigned to one of four groups (n = 9 per group): a) sham-operated group; b) sham-operated group pretreated with sodium tungstate (xanthine oxidase inactivator); c) aorta occlusion group; and d) aorta occlusion group pretreated with sodium tungstate. Descending thoracic aorta occlusion was maintained for 40 mins with a 4-Fr Fogarty embolectomy catheter, followed by 2 hrs of reperfusion. MEASUREMENTS AND MAIN RESULTS Gastric tonometry was performed after completion of the surgical preparation (30-min equilibration) and at 30, 60, 90, and 120 mins of reperfusion. Plasma alanine aminotransferase activity was determined at 120 mins of reperfusion to assess hepatic injury. Bronchoalveolar lavage of the right lung was performed after 120 mins of reperfusion, and the protein content was determined as a measure of pulmonary alveolar-capillary membrane compromise. Descending thoracic aorta occlusion resulted in a significant decrease in gastric intramucosal pH as compared with sham-operated rabbits (p < .001). The change in gastric mucosal hydrogen ion concentration was significantly associated with plasma alanine aminotransferase activity (r2 = .48, p < .01) and bronchoalveolar protein content (r2 = .51, p < .01). Xanthine oxidase inactivation significantly improved gastric intramucosal pH after aortic occlusion and reperfusion (p < .001), with a concomitant attenuation of the release of plasma alanine aminotransferase (p < .05) and accumulation of bronchoalveolar protein (p < .05) during reperfusion. CONCLUSIONS Gastric intramucosal pH was significantly decreased after hepatoenteric ischemia-reperfusion. Furthermore, an increase in gastric intramucosal hydrogen ion concentration was associated with a concomitant increase in tissue injury, a presumed harbinger of multiple organ failure. Gastric intramucosal pH values improved during reperfusion after xanthine oxidase inactivation, concomitant with attenuation of hepatic and pulmonary injury. Gastric tonometry is an important clinical tool that can provide critical insight into the pathogenesis of multiple organ injury after hepatoenteric ischemia-reperfusion. Gastric tonometry may aid in the rapid assessment of pharmacologic interventions designed to attenuate multiple organ injury in similar clinical settings (e.g., trauma, shock, major vascular surgery).

Carbon monoxide releasing molecule-2 increases the velocity of thrombus growth and strength in human plasma

Carbon monoxide derived from degradation of heme by heme oxygenase or carbon monoxide releasing molecules (CORMs) has been demonstrated to decrease thrombosis in vivo and to weakly inhibit platelet aggregation. We tested the hypothesis that carbon monoxide released from tricarbonyldichlororuthenium (II) dimer (CORM-2) would diminish the velocity of formation and strength of plasma thrombi as determined by thrombelastography. Normal plasma was exposed to 0 or 100 μmol/l CORM-2 or inactivated CORM-2 (iCORM-2), with coagulation initiated with tissue factor or celite (n = 8 per condition). Additional experiments utilized factor XIII (FXIII) deficient plasma activated with celite. Coagulation kinetics was monitored with thrombelastography for 15 min. CORM-2, and to a lesser extent, iCORM-2, significantly (P < 0.05) increased the velocity of formation (122 and 56%, respectively) and strength (66 and 57%, respectively) of plasma clots initiated with either tissue factor or celite compared with thrombi not exposed to CORM-2 or iCORM. In FXIII deficient plasma CORM-2 significantly increased the velocity of clot formation (264%) and strength (240%). Carbon monoxide and iCORM-2 derived from CORM-2 markedly enhance the velocity of clot growth and strength. These findings serve as the rationale for further investigations to determine if CORMs could be utilized as hemostatic agents.