Rapamycin up-regulation of autophagy reduces infarct size and improves outcomes in both permanent MCAL, and embolic MCAO, murine models of stroke.

BACKGROUND AND PURPOSE: The role of autophagy in response to ischemic stroke has been confusing with reports that both enhancement and inhibition of autophagy decrease infarct size and improve post-stroke outcomes. We sought to clarify this by comparing pharmacologic modulation of autophagy in two clinically relevant murine models of stroke. METHODS: We used rapamycin to induce autophagy, and chloroquine to block completion of autophagy, by treating mice immediately after stroke and at 24 hours post-stroke in two different models; permanent Middle Cerebral Artery Ligation (MCAL), which does not allow for reperfusion of distal trunk of middle cerebral artery, and Embolic Clot Middle Cerebral Artery Occlusion (eMCAO) which allows for a slow reperfusion similar to that seen in most human stroke patients. Outcome measures at 48 hours post-stroke included infarct size analysis, behavioral assessment using Bederson neurological scoring, and survival. RESULTS: Chloroquine treatment reduced the lesion size by approximately 30% and was significant only in the eMCAO model, where it also improved the neurological score, but did not increase survival. Rapamycin reduced lesion size by 44% and 50% in the MCAL and eMCAO models, respectively. Rapamycin also improved the neurological score to a greater degree than chloroquine and improved survival. CONCLUSIONS: While both inhibition and enhancement of autophagy by pharmacological intervention decreased lesion size and improved neurological scores, the enhancement with rapamycin showed a greater degree of improvement in outcomes as well as in survival. The protective action seen with chloroquine may be in part due to off-target effects on apoptosis separate from blocking lysosomal activity in autophagy. We conclude pharmacologic induction of autophagy is more advantageous than its blockade in physiologically-relevant permanent and slow reperfusion stroke models.

Embolic Stroke Diagnosed by Elevated D-Dimer in a Patient With Negative TEE for Cardioembolic Source.

We report a case of cerebrovascular accident with thromboembolic stroke etiology in a patient who had atrial flutter and negative transesophageal echocardiography (TEE) results. The increased D-dimer levels (1877 ng/mL) initiated referral for magnetic resonance imaging and magnetic resonance angiography of the brain that showed classic recanalization of an embolic thrombus in the angular branch of the left middle cerebral distribution. The D-dimer level of this patient was normalized after 3 months of anticoagulation therapy. Although TEE is considered the gold standard for evaluation of cardiac source of embolism, exclusion of intracardiac thrombus with TEE alone does not eliminate the risk of thromboembolic events. This case highlights the utility of D-dimer as a potential adjunct in the decision-making process to guide investigation of thromboembolism, determine subsequent therapy, and hence reduce the risk of embolic stroke recurrence.

Detection and treatment of intravascular thrombi with magnetofluorescent nanoparticles.

Thrombosis, the formation of a clot within a blood vessel, underlies a number of life-threatening cardiovascular disorders such as heart attack, ischemic stroke, pulmonary embolism, and deep vein thrombosis. These conditions affect the lives of millions of people worldwide and result in significant morbidity and mortality. It is thus crucial to develop novel methodologies to enhance the detection and treatment of these disorders. Thrombolysis, or the dissolution of blood clots, relies upon the administration of exogenous plasminogen activators (PAs) that lyse fibrin. Yet, there are several drawbacks to the use of current PAs, including significant risks of uncontrolled bleeding and suboptimal efficacy and pharmacokinetics. Nanomaterials are well positioned to address these priority issues in thrombolysis, via the alteration of PA pharmacokinetics and biodistribution. Additionally, due to the multifunctional nature of nanoparticles, these thrombolytics may be targeted to the site of occlusion, effectively concentrating the drug where it is most needed. Herein, we describe the methodology associated with the synthesis of a novel thrombus-targeted fibrinolytic nanoagent. At each step of the synthesis, we analyze the nanomaterials, including their physical properties and their ability to bind to thrombosis targets of interest. The effect of the conjugation of tPA to the nanoparticle surface on the amidolytic and fibrinolytic activities of nanoagents is also investigated. Lastly, the in vivo binding of the targeted thrombolytic to intravascular thrombi is examined.

Multifunctional nanoagent for thrombus-targeted fibrinolytic therapy.

BACKGROUND: Current thrombolytic therapies utilize exogenous plasminogen activators (PAs) to effectively lyse clots, restoring blood flow, and preventing tissue and organ death. These PAs may also impair normal hemostasis, leading to life-threatening bleeding, including intracerebral hemorrhage. AIMS: This study aims to develop new thrombus-targeted fibrinolytic agents that harness the multifunctional theranostic capabilities of nanomaterials, potentially allowing for the generation of efficacious thrombolytics while minimizing deleterious side effects. MATERIALS & METHODS: A thrombus-targeted nano-fibrinolytic agent was synthesized using a magnetofluorescent crosslinked dextran-coated iron oxide nanoparticle platform that was conjugated to recombinant tissue PA (tPA). Thrombus-targeting was achieved by derivatizing the nanoparticle with an activated factor XIII (FXIIIa)-sensitive peptide. Human plasma clot binding ability of the targeted and control agents was assessed by fluorescence reflectance imaging. Next, the in vitro enzymatic activity of the agents was assessed by S2288-based amidolytic activity, and an ELISA D-dimer assay for fibrinolysis. In vivo targeting of the nanoagent was next examined by intravital fluorescence microscopy of murine arterial and venous thrombosis. The fibrinolytic activity of the targeted nanoagent compared to free tPA was then evaluated in vivo in murine pulmonary embolism. RESULTS: In vitro, the targeted thrombolytic nanoagent demonstrated superior binding to fresh-frozen plasma clots compared to control nanoagents (analysis of variance, p < 0.05). When normalized by S2288-based amidolytic activity, targeted, control and free tPA samples demonstrated equivalent in vitro fibrinolytic activity against human plasma clots, as determined by ELISA D-dimer assays. The FXIIIa targeted fibrinolytic nanoagent efficiently bound the margin of intravascular thrombi as detected by intravital fluorescence microscopy. In in vivo fibrinolysis studies the FXIIIa-targeted agent lysed pulmonary emboli with similar efficacy as free tPA (p > 0.05). CONCLUSION: The applicability of a FXIIIa-targeted thrombolytic nanoagent in the treatment of thromboembolism was demonstrated in vitro and in vivo. Future studies are planned to investigate the safety profile and overall efficacy of this class of nanoagents.

Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke.

BACKGROUND: Minocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans. METHODS: Five groups of mice were subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females. They were treated with phosphate saline (vehicle) or minocycline (6 mg/kg) immediately after stroke onset. Behavioral outcomes, infarct volumes and cerebral blood flow were assessed. The effect of minocycline on expression and activity of MMP-9 was analyzed. RESULTS: The model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P < 0.0001) and also improved neurologic score (P < 0.0001) in all groups. Moreover, minocycline treatment significantly reduced mortality at 24 hours post stroke (P = 0.037) for aged mice (25% versus 54%). Stroke up-regulated MMP-9 level in the brain, and acute minocycline treatment reduced its expression in both genders (P < 0.0001). CONCLUSION: In a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.

Minocycline and tissue-type plasminogen activator for stroke: assessment of interaction potential.

BACKGROUND AND PURPOSE: New treatment strategies for acute ischemic stroke must be evaluated in the context of effective reperfusion. Minocycline is a neuroprotective agent that inhibits proteolytic enzymes and therefore could potentially both inactivate the clot lysis effect and decrease the damaging effects of tissue-type plasminogen activator (t-PA). This study aimed to determine the effect of minocycline on t-PA clot lysis and t-PA-induced hemorrhage formation after ischemia. METHODS: Fibrinolytic and amidolytic activities of t-PA were investigated in vitro over a range of clinically relevant minocycline concentrations. A suture occlusion model of 3-hour temporary cerebral ischemia in rats treated with t-PA and 2 different minocycline regimens was used. Blood-brain barrier basal lamina components, matrix metalloproteinases (MMPs), hemorrhage formation, infarct size, edema, and behavior outcome were assessed. RESULTS: Minocycline did not affect t-PA fibrinolysis. However, minocycline treatment at 3 mg/kg IV decreased total protein expression of both MMP-2 (P=0.0034) and MMP-9 (P=0.001 for 92 kDa and P=0.0084 for 87 kDa). It also decreased the incidence of hemorrhage (P=0.019), improved neurologic outcome (P=0.0001 for Bederson score and P=0.0391 for paw grasp test), and appeared to decrease mortality. MMP inhibition was associated with decreased degradation in collagen IV and laminin-alpha1 (P=0.0001). CONCLUSIONS: Combination treatment with minocycline is beneficial in t-PA-treated animals and does not compromise clot lysis. These results also suggest that neurovascular protection by minocycline after stroke may involve direct protection of the blood-brain barrier during thrombolysis with t-PA.

Regulation of nonproteolytic active site formation in plasminogen.

Streptokinase may be less effective at saving lives in patients with heart attacks because it explosively generates plasmin in the bloodstream at sites distant from fibrin clots. We hypothesized that this rapid plasmin generation is due to SK's singular capacity to nonproteolytically generate the active protease SK x Pg*, and we examined whether the kringle domains regulate this process. An SK mutant lacking Ile-1 (deltaIle1-SK) does not form SK x Pg*, although it will form complexes with plasmin that can activate plasminogen. When compared to SK, deltaIle1-SK diminished the generation of plasmin in plasma by more than 30-fold, demonstrating that the formation of SK x Pg* plays an important role in SK activity in the blood. The rate of SK x Pg* formation (measured by an active site titrant) was much slower in Glu-Pg, which contains five kringle domains, than in Pg forms containing one kringle (mini-Pg) or no kringles (micro-Pg). In a similar manner, Streptococcus uberis Pg activator (SUPA), an SK-like molecule, generated SUPA x Pg* much slower with bovine Pg than bovine micro-Pg. The velocity of SK x Pg* formation was regulated by agents that influence the conformation of Pg through interactions with the kringle domains. Chloride ions, which maintain the compact Pg conformation, hindered SK x Pg* formation. In contrast, epsilon-aminocaproic acid, fibrin, and fibrinogen, which induce an extended Pg conformation, accelerated the formation of SK x Pg*. In summary, the explosive generation of plasmin in blood or plasma, which diminishes SK's therapeutic effects, is attributable to the formation of SK x Pg*, and this process is governed by kringle domains.

alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator.

The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.

Coevolutionary patterns in plasminogen activation.

The generation of plasmin by plasminogen (Pg) activators (PAs) is a physiologic process in animals that dissolves blood clots and promotes wound healing, blood vessel growth, and the migration of normal and cancerous cells. Pathogenic bacteria have evolved PAs [e.g., streptokinase (SK) and staphylokinase] that exploit the Pg system to infect animals. Animal PAs have a conserved ability to cleave a wide spectrum of animal Pgs, but the ability of bacterial PAs to cleave different animal Pgs is surprisingly restricted. We show that the spectrum of activity of an archetypal bacterial PA (SK) with animal Pgs can be profoundly altered by mutations that affect intermolecular complementarity at sites that participate in complex formation or substrate binding. Comparative sequence analysis of animal plasmins vs. close structural homologues (trypsin and chymotrypsin) that are not molecular targets for invading bacteria indicates that the sites in plasmin that interact with SK are preferentially targeted for mutation. Conversely, intermolecular contact sites in SKs that activate human Pg are more highly conserved than other loci in the molecule or than the same sites in other SKs that activate non-human Pgs. We propose that active modulation of intermolecular complementarity at sites of contact between SK and Pg may represent a competitive evolutionary strategy in a survival battle, whereby animals seek to evade bacterial invasion, and bacteria endeavor to invade their animal hosts.

Structural elements that govern the substrate specificity of the clot-dissolving enzyme plasmin.

There is remarkable homology between the core structures of plasmin, a fibrin clot-degrading enzyme, and factor D, a complement-activating enzyme, despite markedly different biological functions. We postulated that sequence divergence in the loop structures between these two enzymes mediated the unique substrate and inhibitor interactions of plasmin. Recombinant microplasminogens chimerized with factor D sequences at loops 3, 5, and 7 were cleaved by the plasminogen activator urokinase and developed titratable active sites. Chimerization abolished functional interactions with the plasminogen activator streptokinase but did not block complex formation. The microplasmin chimeras showed enhanced resistance (k(i) decreased up to two to three times) to inactivation of microplasmin by alpha(2)-antiplasmin. Microplasmin chimerization had minimal ( approximately 2 fold) effects on the catalytic efficiency for cleavage of small substrates and did not alter the cleavage of fibrin. However, microplasmin and the microplasmin chimeras showed enhanced abilities to degrade fibrin in plasma clots suspended in human plasma. These studies indicate that loop regions of the protease domain of plasmin are important for interactions with substrates, regulatory molecules, and inhibitors. Because modification of these regions affected substrate and inhibitor interactions, loop chimerization may hold promise for improving the clot dissolving properties of this enzyme.

Chimerism reveals a role for the streptokinase Beta -domain in nonproteolytic active site formation, substrate, and inhibitor interactions.

Streptokinase (SK) and staphylokinase form cofactor-enzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K(m) = 16.8 versus 15.2 microm(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.