Use of recombinant activated factor VII in Jehovah's Witness patients with critical bleeding.

INTRODUCTION: The Australian and New Zealand Haemostasis Registry (ANZHR) included patients who received off-licence recombinant activated factor VII (rFVIIa) for critical bleeding from 2000 to 2009. Approximately 1.3% of the ANZHR patients were Jehovah's Witnesses (JWs). We compared them with the non-JW patients in the registry. METHODS: Patient characteristics (e.g. gender, context of bleeding), factors influencing rFVIIa use (e.g. body temperature and pH) and outcomes (e.g. bleeding response (stopped/attenuated or unchanged) to rFVIIa, mortality) were compared between JW and non-JW patients using Fisher's exact chi-square tests and Kruskal-Wallis tests. RESULTS: A total of 42 JW and 3134 non-JW patients were included in the analysis. Approximately 99% (n = 3098) of non-JWs received blood products compared with only 30% (n = 13) of JWs (P < 0.01). The distribution of gender and contexts of critical bleeding in the two groups was significantly different. Approximately 17% of the non-JW patients were hypothermic (T < 35°C) and about 19% were acidotic (pH < 7.2) at the time of initial rFVIIa administration. Conversely, none of the JWs were hypothermic and only one was acidotic. The proportion of positive responders to rFVIIa (stopped/attenuated bleeding following rFVIIa use) was similar in both groups (75% non-JWs, 74% JWs; P = 1.0). Approximately 28% of non-JW and 17% of JW patients were deceased by day 28 following rFVIIa use (P = 0.16). DISCUSSION: Several factors were observed to be significantly different between JW and non-JW patients, yet the proportions of responders to rFVIIa were similar in both groups. The actual factors influencing response to rFVIIa are yet to be determined.

Use of recombinant factor VIIa in patients with amniotic fluid embolism: a systematic review of case reports.

BACKGROUND: Patients with amniotic fluid embolism (AFE) (major cardiac and pulmonary symptoms plus consumptive coagulopathy) have high circulating tissue factor concentrations. Recombinant factor VIIa (rVIIa) has been used to treat hemorrhage in AFE patients even though rVIIa can combine with circulating tissue factor and form intravascular clots. A systematic review was done of case reports from 2003 to 2009 of AFE patients with massive hemorrhage who were and were not treated with rVIIa to assess the thrombotic complication risk. METHODS: MEDLINE was searched for case reports of AFE patients receiving rVIIa (rVIIa cases) and of AFE patients who received surgery to control bleeding but no rVIIa (cohorts who did not receive rVIIa). Additional AFE case reports were obtained from the Food and Drug Administration, the Australian and New Zealand Haemostasis Registry, and scientific meeting abstracts. The risk of a negative outcome (permanent disability or death) in rVIIa cases versus cohorts who did not receive rVIIa was calculated using risk ratio and 95% confidence interval. RESULTS: Sixteen rVIIa cases and 28 cohorts were identified who did not receive rVIIa. All patients had surgery to control bleeding. Death, permanent disability, and full recovery occurred in 8, 6, and 2 rVIIa cases and 7, 4, and 17 cohorts who did not receive rVIIa (risk ratio 2.2, 95% CI 1.4-3.7 for death or permanent disability vs. full recovery). CONCLUSION: Recombinant factor VIIa cases had significantly worse outcomes than cohorts who did not receive rVIIa. It is recommended that rVIIa be used in AFE patients only when the hemorrhage cannot be stopped by massive blood component replacement.

Reduced L-arginine transport contributes to the pathogenesis of myocardial ischemia-reperfusion injury.

Myocardial injury due to ischemia-reperfusion (I-R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I-R process induces a state of insufficient L-arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I-R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia-reoxygenation significantly decreased L-arginine uptake and NO production (42 +/- 2% and 71 +/- 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L-arginine transporter, CAT1, and NVCMs supplemented with L-arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia-reoxygenation. In contrast, L-arginine deprived NVCM had significantly worsened responses to hypoxia-reoxygenation. In isolated perfused mouse hearts, L-arginine infusion during reperfusion significantly improved left ventricular function after I-R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L-arginine availability as a key factor in the pathogenesis of I-R injury. Increasing L-arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I-R. Restoration of L-arginine availability may therefore be a valuable strategy to ameliorate I-R injury.

Inhibition of myocardial apoptosis by postconditioning is associated with attenuation of oxidative stress-mediated nuclear factor-kappa B translocation and TNF alpha release.

Oxidative stress-stimulated nuclear factor-kappa B (NF-kappa B) activation has been associated with rapid transcription of TNF-alpha and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) reduces myocardial apoptosis and inhibits translocation of NF-kappa B and release of TNF-alpha secondary to an attenuation of oxidant generation during reperfusion. Anesthetized rats were subjected to 30 min of ischemia and 3 h of reperfusion and divided randomly to Control or Postcon (three cycles of 10-s reperfusion and 10-s reocclusion applied at the onset of reperfusion) group, respectively. Relative to Control, Postcon reduced the plasma malondialdehyde (1.21 +/- 0.08 vs. 0.8 +/- 0.06* microM/mL) and decreased the generation of superoxide radical in area at risk myocardium (dihydroethidium staining). Compared with Control, Postcon also inhibited translocation of NF-kappa B to nuclei (167% +/- 21% vs. 142% +/- 18%*), decreased the level of plasma TNF-alpha (1,994 +/- 447 vs. 667 +/- 130* pg/mL), and inhibited caspase-3 activity (0.57% +/- 0.1% vs. 0.21% +/- 0.1%*). The number of apoptotic cells (percent total nuclei) in ischemic myocardium was reduced (20% +/- 1% vs. 11% +/- 2%*), consistent with reduced appearance of DNA fragmentation. To support whether oxidant generation is important in the triggering of cytokine release and apoptosis, N-acetylcysteine (NAC), a potent antioxidant agent, was administered before ischemia and at reperfusion. Treatment with NAC inhibited superoxide radical generation and decreased plasma malondialdehyde to a comparable level to that in Postcon, concomitant with an inhibition of NF-kappa B expression (42% +/- 8%*) and reduction of release of TNF-alpha (231 +/- 72* pg/mL). Caspase-3 activity (0.33% +/- 0.1%*) and apoptotic cells (12% +/- 1%*) were also comparably reduced by NAC. These data suggest that Postcon attenuates myocardial apoptosis, reduces caspase-3 activity, and is potentially mediated by inhibiting oxidant-activated NF-kappa B-TNF-alpha signaling pathway. *P < 0.05 Postcon and NAC vs. Control.

Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation.

Opioids introduced at reperfusion (R) following ischemia (I) reduce infarct size much like postconditioning, suggesting the hypothesis that postconditioning increases cardiac opioids and activates local opioid receptors. Anesthetized male rats subjected to 30 min regional I and 3 h R were postconditioned with three cycles of 10 s R and 10 s reocclusion at onset of R. Naloxone (NL), its peripherally restricted analog naloxone methiodide, delta-opioid receptor (DOR) antagonist naltrindole (NTI), kappa-opioid receptor antagonist norbinaltorphimine (NorBNI), and mu-opioid receptor (MOR) antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were administered intravenously 5 min before R. The area at risk (AAR) was comparable among groups, and postconditioning reduced infarct size from 57 +/- 2 to 42 +/- 2% (P < 0.05). None of the antagonists alone altered infarct size. All antagonists abrogated postconditioning protection at higher doses. However, blockade of infarct sparing by postconditioning was lost, since tested doses of NL, NTI, NorBNI, and CTAP were lowered. The efficacy of NorBNI declined first at 3.4 micromol/kg, followed sequentially by NTI (1.1), NL (0.37), and CTAP (0.09), suggesting likely MOR and perhaps DOR participation. Representative small, intermediate, and large enkephalins in the AAR were quantified (fmol/mg protein; mean +/- SE). I/R reduced proenkephalin (58 +/- 9 vs. 33 +/- 4; P < 0.05) and sum total of measured enkephalins, including proenkephalin, peptide B, methionine-enkephalin, and methionine-enkephalin-arginine-phenylalanine (139 +/- 17 vs. 104 +/- 7; P < 0.05) compared with shams. Postconditioning increased total enkephalins (89 +/- 8 vs. 135 +/- 5; P < 0.05) largely by increasing proenkephalin (33 +/- 4 vs. 96 +/- 7; P < 0.05). Thus the infarct-sparing effect of postconditioning appeared to involve endogenously activated MORs and possibly DORs, and preservation of enkephalin precursor synthesis in the AAR.

Preconditioning and postconditioning: innate cardioprotection from ischemia-reperfusion injury.

Reperfusion is the definitive treatment to salvage ischemic myocardium from infarction. A primary determinant of infarct size is the duration of ischemia. In myocardium that has not been irreversibly injured by ischemia, reperfusion induces additional injury in the area at risk. The heart has potent innate cardioprotective mechanisms against ischemia-reperfusion that reduce infarct size and other presentations of postischemic injury. Ischemic preconditioning (IPC) applied before the prolonged ischemia exerts the most potent protection observed among known strategies. It has been assumed that IPC exerts protection during ischemia. However, recent data suggest that cardioprotection is also exerted during reperfusion. Postconditioning (PoC), defined as brief intermittent cycles of ischemia alternating with reperfusion applied after the ischemic event, has been shown to reduce infarct size, in some cases equivalent to that observed with IPC. Although there are similarities in mechanisms of cardioprotection by these two interventions, there are key differences that go beyond simply exerting these mechanisms before or after ischemia. A significant limitation of IPC has been the inability to apply this maneuver clinically except in situations where the ischemic event can be predicted. On the other hand, PoC is applied at the point of service in the hospital (cath-lab for percutaneous coronary intervention, coronary artery bypass grafting, and other cardiac surgery) where and when reperfusion is initiated. Initial clinical studies are in agreement with the success and extent to which PoC reduces infarct size and myocardial injury, even in the presence of multiple comorbidities.

Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling.

OBJECTIVE: Using non-selective and selective protein kinase C (PKC) epsilon and delta isoform inhibitors, we tested the hypothesis that the cardioprotective phenotype invoked by postconditioning (postcon) is dependent on PKC signalling. Furthermore, we determined whether postconditioning alters pPKCepsilon and/or pPKCdelta in cytosolic and mitochondrial fractions. METHODS: Male Sprague-Dawley rats underwent 30 min left coronary artery (LCA) occlusion followed by 3 h of reperfusion. Rats were randomised to the following groups: Untreated, no intervention either before or after LCA occlusion; Postcon, 3 cycles of 10-s full reperfusion and 10-s re-occlusion, initiated immediately at the onset of reperfusion; Chelerythrine (non-selective PKC inhibitor, 5 mg/kg)+/-postcon; Rottlerin (PKCdelta inhibitor, 0.3 mg/kg)+/-postcon; KIE1-1 (PKCepsilon inhibitor, 3.8 mg/kg)+/-postcon. A subset of rats was employed to assess pPKCepsilon and/or pPKCdelta in sham, Isch/RP (30-min LCA occlusion followed by 30-min reperfusion), and postcon-treated hearts. RESULTS: Infarct size, expressed as area of necrosis as a percentage of the area at risk, AN/AAR (%), was significantly reduced by postcon compared to control (untreated) rats (39+/-2% vs. 53+/-1% in control, P<0.001). Treatment with chelerythrine alone or the PKCepsilon antagonist KIE1-1 alone at reperfusion had no effect on infarct size compared to control. In contrast, the infarct-sparing effect of postcon was abrogated by non-selective PKC inhibition and PKCepsilon antagonism (50+/-2% and 50+/-1%, respectively, P<0.002). Inhibition of PKCdelta reduced infarct size to values comparable to that in postcon group (36+/-3% vs. 39+/-2%). However, postcon in the presence of PKCdelta inhibitor did not enhance the infarct-sparing effects (38+/-2%). In addition, pPKCepsilon in postcon hearts was significantly higher in the total cell homogenate (10338+/-1627 vs. 4165+/-608 in Isch/RP, arbitrary units), and pPKCdelta translocation to mitochondria was significantly less (>2-fold decrease) compared to Isch/RP. CONCLUSION: These data suggest that postcon modulates PKC during early reperfusion by increasing PKCepsilon expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCdelta to mitochondria and associated deleterious signalling.

Adenosine receptor-mediated coronary vascular protection in post-ischemic mouse heart.

This study evaluated the ability of A1 and A3 adenosine receptor (AR) agonism, and A1, A2A, A2B and A3AR antagonism (revealing "intrinsic" responses), to modify post-ischemic coronary dysfunction in mouse heart. Vascular function was assessed before and after 20 min global ischemia and 30-45 min reperfusion in Langendorff perfused C57/Bl6 mouse hearts. Ischemic insult impaired coronary sensitivity to the endothelial-dependent dilators ADP (pEC50=6.8+/-0.1 vs. 7.6+/-0.1, non-ischemic) and acetylcholine (pEC50=6.1+/-0.1 vs. 7.3+/-0.1 in non-ischemic), and for the mixed endothelial-dependent/independent dilator 2-chloroadenosine (pEC50=7.5+/-0.1 vs. 8.4+/-0.1, non-ischemic). Endothelium-independent dilation in response to nitroprusside was unaltered (pEC50=7.0+/-0.1 vs. 7.1+/-0.1 in non-ischemic). Pre-treatment with a selective A1AR agonist (50 nM CHA) failed to modify coronary dysfunction, whereas A1AR antagonism (200 nM DPCPX) worsened the effects of I/R (2-chloroadenosine pEC50=6.9+/-0.1). Conversely, A3AR agonism (100 nM Cl-IB-MECA) did reduce effects of I/R (pEC50s=8.0+/-0.1 and 7.3+/-0.1 for 2-chloroadenosine and ADP, respectively), whereas antagonism (100 nM MRS1220) was without effect. While A2AAR agonism could not be assessed (due to pronounced vasodilatation), A2AAR antagonism (100 nM SCH58261) was found to exert no effect, and antagonism of A2BARs (50 nM MRS1754) was also ineffective. The protective actions of A3AR agonism were also manifest as improved reactive hyperemic responses. Interestingly, post-ischemic coronary dysfunction was also limited by: Na+-H+ exchange (NHE) inhibition with 10 or 50 microM BIIB-513 (2-chloroadenosine pEC50s=7.8+/-0.1, either dose), an effect not additive with A3AR agonism; Ca2+ antagonism with 0.3 microM verapamil (2-chloroadenosine pEC50=7.9+/-0.1); and Ca2+ desensitization with 5 mM BDM (2-chloroadenosine pEC50=7.8+/-0.1). In contrast, endothelin antagonism (200 nM PD142893) and anti-oxidant therapy (300 microM MPG+150 U/ml SOD+600 U/ml catalase) were ineffective. Our data collectively confirm that ischemia selectively impairs endothelial function and reactive hyperemia independently of blood cells. Vascular injury is intrinsically limited by endogenous (but not exogenous) activation of A1ARs, whereas exogenous A3AR activation further limits dysfunction (improving post-ischemic vasoregulation). Finally, findings suggest this form of post-ischemic coronary injury is unrelated to endothelin or oxidant stress, but may involve modulation of Ca2+ overload and/or related ionic perturbations.

Myocardial protection in reperfusion with postconditioning.

Reperfusion is the definitive treatment for coronary occlusive disease. However, reperfusion carries the potential to exacerbate lethal injury, termed 'reperfusion injury'. Studies have suggested that reperfusion injury events are triggered during the early moments of reflow, and determine, in part, the severity of downstream manifestations of postischemic injury, including endothelial dysfunction, infarction and apoptosis. The application of brief iterative episodes of reflow (reoxygenation) and reocclusion (ischemia, hypoxia) at the immediate onset of reperfusion, which has been termed 'postconditioning' by the authors, reduces many manifestations of postischemic injury, notably infarct size, apoptosis, coronary vascular endothelial injury and reperfusion arrhythmias. Cardioprotection with postconditioning has been reported to be comparable with that observed using the gold standard maneuver ischemic preconditioning. In contrast to preconditioning, which exerts its effects primarily during the index ischemia, postconditioning appears to exert its effects during reperfusion alone. Postconditioning modifies the early phase of reperfusion in ways that are just beginning to be understood. It appears to first: reduce the oxidant burden and consequent oxidant-induced injury; secondly, attenuate the local inflammatory response to reperfusion; and thirdly, engage end effectors and signaling pathways implicated in other cardioprotective maneuvers, such as ischemic and pharmacologic preconditioning. Postconditioning seems to trigger the upregulation of survival kinases principally known to attenuate the pathogenesis of apoptosis and possibly necrosis. The postconditioning phenomenon has been reproduced by a number of independent laboratories and has been observed in both large and small animal in vivo models, as well as in ex vivo and cell culture models. In contrast to preconditioning, postconditioning may have widespread clinical application because it can be applied during reperfusion at the point of service for angioplasty, stenting, cardiac surgery and organ transplantation.

Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine.

OBJECTIVE: This study tested the hypothesis that brief cycles of iterative ischemia-reperfusion at onset of reperfusion (termed "postconditioning", post-con) delays washout of intravascular adenosine and thereby increases endogenous adenosine receptor (AR) activation during the early moments of reperfusion (R). METHODS: Isolated mouse hearts were subjected to 20 min global ischemia (I) and 30 min R with or without post-con (3 or 6 cycles of 10 s R&I). Intravascular purines in coronary effluent were analyzed by HPLC. To assess the functional role of endogenous AR activation in post-con, an open-chest rat model of myocardial infarction was employed. Rats were randomly divided into 11 groups: control, no intervention at R; post-con, three cycles of 10 s R followed by 10 s LCA re-occlusion immediately upon R. In the following interventions, drugs (or vehicle) were administered 5 min before R in the absence or presence (+/-) of post-con. Vehicle (DMSO < 300 microl/kg); 8-SPT (non-selective AR antagonist, 10 mg/kg) +/- post-con; DPCPX (A(1A)R antagonist, 0.1 mg/kg) +/- post-con; ZM241385 (A(2A)AR antagonist, 0.2 mg/kg) +/- post-con; MRS1523 (A(3)AR antagonist, 2 mg/kg) +/- post-con. RESULTS: In isolated mouse hearts, post-con reduced diastolic pressure during both early (26+/-3* vs. 37+/-3 mmHg at 5 min) and late (22+/-3* vs. 34+/-3 mmHg at 30 min) R. Post-con also hastened the early recovery of contractile function (developed pressure 39+/-6* vs. 16+/-2 mmHg at 5 min R), although differences did not persist at 30 min R. Importantly, post-con was associated with reduced adenosine washout (58+/-5* vs. 155+/-16 nM/min/g) at 2 min R suggesting greater retention time of intravascular adenosine. In rats, post-con significantly attenuated infarct size compared to control (40+/-3% vs. 53 +/- 2%* in control), an effect that was unaltered by DPCPX (42 +/- 2%) but was abrogated by 8-SPT (50 +/- 2%), ZM241385 (49 +/- 3%) or MRS1523 (52 +/- 1%) (P < 0.02). CONCLUSION: These data suggest that post-con involves endogenous activation of A(2A) and A3 but not A1AR subtypes. This activation may be linked to the delay in the washout of intravascular adenosine during the early minutes of R during which post-con is applied.

Mediators of coronary reactive hyperaemia in isolated mouse heart.

1. Mechanisms regulating coronary tone under basal conditions and during reactive hyperaemia following transient ischaemia were assessed in isolated mouse hearts. 2. Blockade of NO-synthase (50 muM L-NAME), K(ATP) channels (5 muM glibenclamide), A(2A) adenosine receptors (A(2A)ARs; 100 nM SCH58261), prostanoid synthesis (100 muM indomethacin), and EDHF (100 nM apamin+100 nM charybdotoxin) all reduced basal flow approximately 40%. Effects of L-NAME, glibenclamide, and apamin+charybdotoxin were additive, whereas coadministration of SCH58261 and indomethacin with these inhibitors failed to further limit flow. 3. Substantial hyperaemia was observed after 5-40 s occlusions, with flow increasing to a peak of 48+/-1 ml min(-1) g(-1). Glibenclamide most effectively inhibited peak flows (up to 50%) while L-NAME was ineffective. 4. With longer occlusions (20-40 s), glibenclamide alone was increasingly ineffective, reducing peak flows by approximately 15% after 20 s occlusion, and not altering peak flow after 40 s occlusion. However, cotreatment with L-NAME+glibenclamide inhibited peak hyperaemia by 70 and 25% following 20 and 40 s occlusions, respectively. 5. In contrast to peak flow changes, sustained dilation and flow repayment over 60 s was almost entirely K(ATP) channel and NO dependent (each contributing equally) with all occlusion durations. 6. Antagonism of A(2A)ARs with SCH58261 reduced hyperaemia 20-30% whereas inhibition of prostanoid synthesis was ineffective. Effects of A(2A)AR antagonism were absent in hearts treated with L-NAME and glibenclamide, supporting NO and K(ATP)-channel-dependent effects of A(2A)ARs. 7. EDHF inhibition alone exerted minor effects on hyperaemia and only with longer occlusions. However, residual hyperaemia after 40 s occlusion in hearts treated with L-NAME+glibenclamide+SCH58261+indomethacin was abrogated by cotreatment with apamin+charybdotoxin. 8. Data support a primary role for K(ATP) channels and NO in mediating sustained dilation after coronary occlusion. While K(ATP) channels (and not NO) are also important in mediating initial peak flow adjustments after brief 5-10 s occlusions, their contribution declines with longer 20-40 s occlusions. Intrinsic activation of A(2A)ARs is important in triggering K(ATP) channel/NO-dependent hyperaemia. Synergistic effects of combined inhibitors implicate interplay between mediators, with compensatory changes occurring in K(ATP) channel, NO, and/or EDHF responses when one is individually blocked.