Alpha-fibrinogenases.

Snake venoms contain a number of serine and metalloproteinases, included among these are the fibrinolytic metalloproteinases. When the fibrinolytic enzymes were first isolated from viper venoms it was postulated that there may be a clinical application for these enzymes in the treatment of occlusive thrombi, such as those occurring in the great arteries and veins of cardiac and cerebral circulation as well as peripheral arteries and veins. In the ensuing years a substantial body of literature has been generated on the identification and characterization of the fibrinolytic enzymes from a broad spectrum of snake species. In this report we describe the biological properties and positive clinical features of the class of enzymes known as alpha-fibrinogenases. Fibrolase, a fibrinolytic metalloproteinase originally isolated from Agkistrodon contortrix contortrix venom, is the representative fibrinolytic enzyme used for the description and characterization of the alpha-fibrinogenases in this chapter. The biochemical and physiochemical properties and in vivo activity of the enzyme are described as well as in vitro studies using a platelet avid chimera of fibrolase. The chimera was formed by coupling fibrolase to an Arg-Gly-Asp (RGD) like peptide imparting inhibitory activity on platelet aggregation and thrombus formation, while maintaining full fibrinolytic activity. Fibrolase has also been modified through the adduction of polyethylene glycol to reduce the rate of clearance from the circulation. In this review we also include a description of alfimeprase, a recombinant fibrinolytic enzyme derived from fibrolase, and follow the development of the enzyme as a potential clinical agent in the clearance of occlusive thrombi. Alfimeprase is presently in clinical trials for two indications: the treatment of peripheral arterial occlusions (in which phase II is nearing successful completion), and for use in the clearance of occluded vascular access catheters in direct competition with plasminogen activators.

Estrogen receptor-alpha populations change with age in commercial laying hens.

Older hens in production lay larger but fewer eggs than younger birds, and the incidence of soft and broken shells is greater in older hens than younger. These changes are attributable at least in part to changing hormone profiles and diminished ability of the hen to transport calcium at the duodenum. In further exploration of this relationship, a study was conducted with three ages of Hy-Line W-36 birds: prelay pullets (PL; 19 wk, 0% production), peak-production hens (PP; 29 wk, approximately 93% production), and late-stage hens (LS; 71 wk, approximately 80% production). Hens from the PP and LS groups were palpated for presence of an egg in the shell gland; hens were then euthanized and tissues (kidney, shell gland, hypothalamus) were removed for quantification of estrogen receptor-alpha (ERalpha) populations via immunocytochemical and Western blot analyses. Localization of ERalpha by immunostaining in the shell gland showed differences among age groups; however, no differences were noted in localization of ERalpha between age groups in the kidney and hypothalamus. In both the kidney and the shell gland there was a decrease in the amount of ERalpha, as detected by immunoblotting, in the LS hens compared to PL and PP birds (P < 0.05). The results suggest that failure of calcium regulating mechanisms with age may be mediated at least in part through the reduced populations of estrogen receptors in certain critical tissues.

New directions in thrombolytic therapy.

Although current thrombolytic agents have proven their clinical benefit, the failure to rapidly reperfuse some patients and the persistent bleeding risk represent areas for improvement in therapy. In the past two years, the field has been advanced by the regulatory approval of agents with greater ease of administration, continued development of new agents and exploration of the use of more advanced antiplatelet therapies in combination with thrombolytic agents. Finally, a new class of directly acting fibrinolytic agents is available.

Alfimeprase: pharmacology of a novel fibrinolytic metalloproteinase for thrombolysis.

Alfimeprase is a recombinantly produced, truncated form of fibrolase, a known directly fibrinolytic zinc metalloproteinase that was first isolated from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Both fibrolase and alfimeprase have been shown to have direct proteolytic activity against the fibrinogen Aalpha chain. In vivo pharmacology studies have shown that thrombolysis with alfimeprase is up to 6 times more rapid than with plasminogen activators. Alfimeprase can be bound and neutralized by serum alpha(2)-macroglobulin, a prevalent mammalian protease inhibitor which is capable of forming a macromolecular complex with alfimeprase. As a result, systemic bleeding complications have been greatly reduced due to the inhibitory effects of alpha(2)-macroglobulin. This article reviews the biochemical in vitro and in vivo characteristics of this novel acting thrombolytic.

Evidence of a specific pancreatic polypeptide receptor in rat arterial smooth muscle.

Pancreatic polypeptide (PP) is a member of the PP fold family of regulatory peptides. Studies have shown that neuropeptide Y, peptide YY, and PP increased gastrointestinal motility. The GI effects of neuropeptide Y and peptide YY were accompanied by an increase in mean arterial blood pressure; however, PP decreased mean arterial blood pressure. Cloning of a receptor of the neuropeptide Y family with high affinity for PP has been reported. This Y4 receptor is present in intestine, pancreas, and prostate, and its mRNA has been detected in brain and coronary artery. We found in vitro evidence of PP-mediated inhibition of arterial neurogenic vasoconstriction. We have also detected Y4 mRNA in rat peripheral arteries. These findings suggest a potential role for the Y4 receptor in regulating vascular tone.

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice.

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

Recombinant human megakaryocyte growth and development factor attenuates postbypass thrombocytopenia.

BACKGROUND: Cardiopulmonary bypass contributes to platelet loss and dysfunction by exposure to shear stresses, foreign surfaces, and hypothermia. This study tested the hypothesis that pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) accelerates recovery of the platelet population after hypothermic extracorporeal circulation (HEC). METHODS: In a blinded study, subcutaneous injections of drug or placebo were given to dogs daily for 3 days preoperatively (day 0, 1, and 2) with no drug on day 3. On day 4, the animal was prepared for arteriovenous HEC. After heparinization, HEC was initiated at 30 to 40 mL x kg(-1) x min(-1). Hypothermic extracorporeal circulation (25 degrees C) was continued for 90 minutes. RESULTS: Preoperative platelet count (x10(3) platelets/microL) did not differ from predrug count in placebo (256+/-27 versus 255+/-20) or PEG-rHuMGDF (271+/-30 versus 291+/-38). During 60 minutes of HEC, the platelet count decreased to approximately 10% of baseline in placebo (29+/-5) and PEG-rHuMGDF (46+/-8), and recovered to approximately 70% of baseline after rewarming (90 minutes of HEC: placebo, 185+/-17, versus PEG-rHuMGDF, 169+/-22). After HEC, platelet count was greater in PEG-rHuMGDF-treated animals (p < 0.05) without altering function (aggregation responses). Within the first 6 hours after HEC, platelet count in PEG-rHuMGDF-treated animals was rising and increased to 260+/-29 (p < 0.01), but was unchanged in placebo animals (186+/-21). Thereafter, platelet count in placebo animals declined to a nadir of 124+/-15 (72 hours after HEC), whereas platelet count in PEG-rHuMGDF animals approximated the preoperative value (>200) at all times. CONCLUSIONS: Appropriately timed presurgical administration of PEG-rHuMGDF counteracts post-HEC relative thrombocytopenia without increasing platelet population and enhancing aggregation preoperatively or during extracorporeal circulation.

BIBP3226 inhibits neuropeptide Y and pancreatic polypeptide potentiated neurogenic vasoconstriction.

Neuropeptide Y (NPY) potentiates the contractile response of the rat caudal artery to adrenergic nerve stimulation in-vitro. The NPY Y1 selective antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininami de), inhibited the vascular effects of NPY in rat caudal artery preparations in-vitro (IC50 =126 nM). BIBP3226 also inhibited the effects of the selective Y1 agonist [Leu31,Pro34]NPY and completely abolished the effects of avian pancreatic polypeptide that was shown to be capable of potentiating neurogenic vasoconstriction in this preparation. These effects were reversible and are most likely mediated by the Y1 receptor subtype since we failed to observe any functional evidence of a Y2 receptor subtype in rat caudal artery. The caudal artery provides a useful functional assay for pharmacological analysis of NPY and NPY antagonists.

The effect of MGDF on platelet function and thrombosis in animal models.

Recombinant human megakaryocyte growth and development factor (rHuMGDF) is a recombinant form of the endogenous c-mpl ligand, thrombopoietin (TPO). rHuMGDF (and c-mpl ligands in general) can produce a measurable sensitization of platelets to known platelet agonists. Our laboratory has observed this sensitization in vitro in both platelet-rich plasma and whole blood and ex vivo in platelets from animals receiving rHuMGDF. Concurrently, clear increases in the tyrosine phosphorylation of Jak2 and c-mpl receptor can be observed both in vitro and ex vivo. To assess the in vivo prothrombotic potential of rHuMGDF, a rabbit carotid artery model of cyclic flow reduction (CFR) was used. Intravenous administration of platelet-sensitizing dosages of rHuMGDF had no effect on the CFR pattern, whereas control experiments demonstrated that the CFR pattern can be modulated by both platelet sensitizing (epinephrine) and antithrombotic (aspirin and ketanserin) agents. We conclude that thrombopoiesis can be observed at dosages that do not sensitize platelets, and further, that platelet-sensitizing dosages of rHuMGDF do not necessarily enhance platelet-dependent thrombosis in this model.

Tryptase mediates hyperresponsiveness in isolated guinea pig bronchi.

Hyperresponsiveness of airway smooth muscle to allergens and environmental factors has long been associated with the pathophysiology of asthma. Tryptase, a serine protease of lung mast cells, has been implicated as one of the mediators involved in the induction of hyperresponsiveness. As a consequence, tryptase inhibitors have become the subject of study as potential novel therapeutic agents for asthma. Secretory leukocyte protease inhibitor (SLPI) is a naturally occurring protein of human airways which exhibits anti-tryptase activity. To assess the potential therapeutic utility of SLPI in asthma, its effects were evaluated using in vitro and ex vivo models of airway hyperresponsiveness and compared with the effects of the small molecule tryptase inhibitor APC-366. Our results demonstrate that SLPI inhibits tryptase-mediated hyperresponsiveness in vitro and attenuates the hyperresponsiveness observed in airway smooth muscle from antigen-sensitized animals subjected to antigen exposure. The small molecule tryptase inhibitor APC-366 has a similar inhibitory effect. Thus, tryptase appears to be a significant contributor to the development of hyperresponsiveness in these models. To the extent that tryptase contributes to the development and progression of asthma, SLPI may possess therapeutic potential in this disease setting.

Defective platelet activation in G alpha(q)-deficient mice.

Platelets are small disc-shaped cell fragments which undergo a rapid transformation when they encounter vascular damage. They become more spherical and extrude pseudopodia, their fibrinogen receptors are activated, causing them to aggregate, they release their granule contents, and eventually form a plug which is responsible for primary haemostasis. Activation of platelets is also implicated in the pathogenesis of unstable angina, myocardial infarction and stroke. Here we show that platelets from mice deficient in the alpha-subunit of the heterotrimeric guanine-nucleotide-binding protein Gq are unresponsive to a variety of physiological platelet activators. As a result, G alpha(q)-deficient mice have increased bleeding times and are protected from collagen and adrenaline-induced thromboembolism. We conclude that G alpha(q) is essential for the signalling processes used by different platelet activators and that it cannot be replaced by G alpha(i) or the beta gamma subunits of the heterotrimeric G proteins. G alpha(q) may thus be a new target for drugs designed to block the activation of platelets.

Transgenic mice overexpressing human c-mpl ligand exhibit chronic thrombocytosis and display enhanced recovery from 5-fluorouracil or antiplatelet serum treatment.

The consequences of long-term in vivo expression of human c-mpl ligand in a mouse model were examined. Transgenic mice expressing the human full-length cDNA in the liver exhibited a fourfold increase in circulating platelet count that persisted stably over the life of the animals. Transgenic animals thrived and appeared healthy for at least 500 days. Transgenic platelets appeared normal with respect to surface antigens and response to platelet aggregation agonists. The highest-expressing transgenic line maintained human c-mpl ligand serum levels of 3 ng/mL. Megakaryocyte numbers in bone marrow and spleen were elevated, as were bone marrow and spleen megakaryocyte colony-forming cells (MEG-CFC). Megakaryocytes were observed in the bone marrow, spleen, liver, and lung, but in no other sites. Circulating myeloid and lymphoid cell populations were increased twofold. Additionally, the animals had a slight but significant anemia despite an increase in marrow colony-forming units-erythroid (CFU-E). No evidence of myelofibrosis was observed in the bone marrow. The platelet nadir in response to administration of either antiplatelet serum (APS) or 5-fluorouracil (5FU) was significantly reduced relative to the control level. Furthermore, the red blood cell (RBC) nadir was reduced relative to control levels in both models, suggesting that c-mpl ligand can directly or indirectly support the maintenance of erythrocyte levels following thrombopoietic insult.

Reduction in infarct size by ischemic preconditioning persists in a chronic rat model of myocardial ischemia-reperfusion injury.

Ischemic preconditioning (PC) has been consistently observed to reduce infarct size in models of regional myocardial ischemia. However, it is also known to render the heart resistant to injury for only a finite period of time (< 2 h). Myocardial adenosine is widely believed to be one of the mediators of PC and may produce myoprotection in part through an anti-neutrophil effect during the early reperfusion period. When infarct size is assessed following a relatively short period of reperfusion (< 3 h) PC hearts may appear protected although reperfusion injury in the myocardium may be ongoing. Thus, infarct expansion may occur as the effects of PC fade. To substantiate that PC produces a sustained reduction in myocardial necrosis, 27 male Sprague-Dawley rats were anesthetized with pentobarbital and instrumented for regional coronary occlusion (30 min) and reperfusion (7 days). Animals were randomized to a control group (n = 16) or PC (n = 11), which consisted of 2 cycles of 5 min of ischemia and 5 min of reperfusion immediately prior to coronary occlusion. Successful reperfusion was confirmed visually and the occluding suture was left in the chest during recovery. Seven days later, staining for risk area was made by the injection of Evans blue dye while the occluder was in place and necrosis was detected with triphenyltetrazolium chloride staining. Planimetry was performed by a blinded investigator who found the risk area to be 27.2 +/- 1.6 and 33.6 +/- 1.7% of the left ventricle (p = NS) in PC and controls, respectively. All hemodynamic measurements were comparable between groups at all times during ischemia and reperfusion. PC reduced infarct size from 43.3 +/- 2.0% of area at risk to 20.6 +/- -2.1%, a 48% reduction (p < 0.01), and eliminated transmural necrosis which was common in the control group. From these studies we conclude that ischemic PC results in a permanent reduction in infarct size rather than a transient reduction in infarct size in the context of a gradually evolving infarction due to reperfusion injury.

Pretreatment with a blocking monoclonal antibody to P-selectin accelerates pharmacological thrombolysis in a primate model of arterial thrombosis.

It has been established that the fibrin content of a developing thrombus can be dramatically reduced with the use of the GA6 monoclonal antibody, which is directed against P-selectin (CD62p). This effect is probably related to diminished tissue factor activity on monocytes in the presence of P-selectin antagonism. Therefore, we hypothesized that an occlusive arterial thrombus formed in the presence of a P-selectin monoclonal antibody would be more susceptible to lysis with standard thrombolytic therapy. To test this hypothesis, 22 male cynomolgus monkeys were anesthetized and instrumented for induction of thrombosis of a femoral artery. Endothelial injury was induced by passing a 150-microA anodal current through a small electrode that was placed in the femoral artery. Blood flow through the artery was continuously monitored using an ultrasonic transit-time flowmeter. The GA6 monoclonal antibody (1 mg/kg) or control, isotype matched mouse IgG1 (P23 or P7) was administered i.v. 1 hr before electrolytic endothelial injury. In the P23 group (n = 11), an occlusive thrombus formed in 52.1 +/- 8.5 min, and in the GA6 group (n = 11), an occlusive thrombus formed in an average time of 52.0 +/- 8.1 min. After formation of an occlusive thrombus, the current was terminated and intravenous heparin (100 U/kg + 50 U/kg/hr) was administered to prevent clot extension.(ABSTRACT TRUNCATED AT 250 WORDS)

Megakaryocyte growth and development factor (MGDF) moderately enhances in-vitro platelet aggregation.

Megakaryocyte growth and development factor (MGDF) is a novel cytokine which promotes the development of immature megakaryocytes into platelets. We tested the hypothesis that MGDF would alter the sensitivity of platelets to aggregating agents as assessed by in-vitro platelet aggregometry. Platelet aggregation in the presence or absence of MGDF was tested with single doses of clinically relevant aggregating agents. A dose-dependent enhancement of the aggregation response to epinephrine was noted in MGDF treated platelets. When a range of concentrations of ADP were used to generate an aggregation dose-response curve, the addition of MGDF to platelet rich plasma shifted the dose response curve to the left. The effect of MGDF on platelet aggregation was partially prevented by the coincubation of platelets with a soluble form of the receptor for MGDF, the extracellular domain of c-mpl. In addition, we demonstrate that exogenous MGDF is able to induce tyrosine phosphorylation of platelet proteins with apparent molecular weights of 85 kDa and 130 kDa. From these data we conclude that exogenously added MGDF moderately increases the sensitivity of platelets to aggregating agents through a mechanism which appears to involve tyrosine phosphorylation of platelet proteins.

Comparative effects of the potassium channel openers cromakalim and pinacidil and the cromakalim analog U-89232 on isolated vascular and cardiac tissue.

ATP-sensitive potassium (K+ATP) channel openers such as cromakalim and pinacidil exhibit both potent vasodilatory and anti-ischemic properties. U-89232, a cyanoguanidine analog of cromakalim, has recently been found to exhibit myocardial protection during ischemia without altering in vivo hemodynamics. We examined the effects of U-89232, cromakalim and pinacidil in isolated vascular and cardiac tissue and tested whether glyburide, a KATP channel blocker, could antagonize their effects. All three compounds produced concentration-dependent relaxation in isolated vascular segments, with cromakalim being approximately 100-fold more potent than either pinacidil or U-89232. Glyburide completely antagonized the effects of pinacidil but merely blunted the effects of cromakalim and U-89232. In an isolated rabbit cardiac tissue preparation, U-89232 had little effect on maximum tension in cardiac muscle, whereas cromakalim and pinacidil significantly decreased maximum developed tension in a concentration-dependent manner. Glyburide effectively antagonized the effects of cromakalim and pinacidil in cardiac tissue. These data suggest that U-89232, although chemically related to cromakalim, possesses activity which is not common to known potassium channel openers.

Cardioprotection with U-89232 is not reversible with glibenclamide: evidence of a novel anti-ischemic agent derived from cromakalim.

We have previously reported that cromakalim and U-89232 reduce infarct size in a rabbit model of myocardial ischemia. Because U-89232 appeared to lack activity in the vasculature, we tested its reversibility with glibenclamide. Twenty-eight ketamine-xylazine anesthetized open-chest, New Zealand White rabbits were instrumented for regional coronary occlusion and reperfusion. Study animals received either cromakalim, U-89232 or vehicle. In some animals, glibenclamide was administered. All animals were then subjected to ischemia (30 min) and reperfusion (120 min), and necrosis was determined using tetrazolium. With comparable hemodynamics and myocardium at risk, infarct size in control animals was 35.5 +/- 4.6% of risk region, and was not different from glibenclamide-treated animals (37.7 +/- 5.8%). Cromakalim alone has been shown to be protective, however when combined with glibenclamide necrosis amounted to 35.1 +/- 3.8% of the risk region (p = NS vs. control). In contrast, U-89232 was protective in the presence of glibenclamide (17.2 +/- 4.9% of the risk region). We conclude that U-89232 produces myoprotection independent of K-ATP channel inhibition, indicating that this compound possesses novel anti-ischemic characteristics.

Induction of neointimal hyperplasia by coronary angioplasty balloon overinflation: comparison of feeder pigs to Yucatan minipigs.

We evaluated the use of simple balloon overinflation to induce neointimal hyperplasia in a porcine model of coronary artery restenosis. By using standard percutaneous transluminal coronary angioplasty techniques, left anterior descending (LAD) and/or left circumflex (LCX) coronary arteries of either juvenile feeder pigs or adult Yucatan minipigs were intentionally overinflated. Four weeks later, resultant neointimal hyperplastic responses (neointima/media area; NI/M) were quantitated morphometrically. At all ballooned sites neointimal hyperplasia occurred only when the internal elastic lamina (IEL) was ruptured; the degree of hyperplasia correlated directly with the injury index, that is, the percentage of IEL circumference that fractured (r = 0.74; n = 25; p < 0.05). Despite similar injury indexes in the LAD bed, there was a trend (p = 0.07; analysis of variance) toward greater NI/M ratios in the Yucatan minipig versus the feeder pig group (1.14 +/- 0.21 vs 0.73 +/- 0.09, n = 7/group). We found no such trend in the LCX bed, where the injury index (25.7% +/- 3.5%) was significantly greater than that of the LAD (18.2% +/- 1.2%, p < 0.05). If variations in balloon-induced vascular injury are accounted for, the technique of balloon overinflation of coronary arteries should prove useful in testing potential antirestenotic agents in either adult or juvenile pigs.

Ischemic preconditioning fails to limit infarct size in reserpinized rabbit myocardium. Implication of norepinephrine release in the preconditioning effect.

BACKGROUND: Infarct size reduction by ischemic preconditioning is believed to be mediated by adenosine; however, whether adenosine is the factor responsible for the initiation of this protection remains unknown. It is possible that during preconditioning, adenosine stimulates receptors on presynaptic nerve terminals and retards the release of norepinephrine (NE) during the prolonged ischemia or that NE release during preconditioning augments adenosine production. METHODS AND RESULTS: To test whether the release of NE is involved in the preconditioning phenomenon, rabbits were pretreated with reserpine (5 mg/kg sc, 24 hours before) to deplete presynaptic nerve terminals of NE stores. On the day of the experiment, the rabbits were anesthetized with ketamine-xylazine and instrumented for coronary occlusion. Nonreserpinized animals were used as controls. The control group (n = 7) was subjected to 30 minutes of coronary occlusion and 120 minutes of reperfusion (ischemia-reperfusion) only. The preconditioned group (n = 10) received 5 minutes of preconditioning ischemia and 10 minutes of reperfusion before the prolonged ischemia-reperfusion. Of the reserpinized animals, half (n = 7) received preconditioning before ischemia-reperfusion and the remaining animals (n = 7) did not. At termination of the experiment, an intravenous tyramine challenge (1 mg/kg) was used to confirm NE depletion in reserpinized rabbits. The resulting infarcts were measured with tetrazolium and planimetry. With comparable hemodynamics and areas at risk, infarct size in control animals was 39.8 +/- 2.1% of the risk region. Preconditioned animals showed an expected reduction of infarct size to 14.8 +/- 2.2% of risk region (P < .05 vs control). Of the reserpinized animals, those that received reserpine alone had infarcts that were 38.5 +/- 4.5% of risk region, and those that were preconditioned had infarcts that were 41.4 +/- 3.6% of risk region, which was not significantly different than the control group. CONCLUSIONS: We conclude that preconditioning fails to protect ischemic-reperfused myocardium in reserpinized rabbit myocardium, indicating that the release of NE during either preconditioning or prolonged ischemia is critical to preconditioning mediated protection.