Irina Y. Sazonova

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-α1 (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.

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.

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

BackgroundMinocycline 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.MethodsFive 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.ResultsThe 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).ConclusionIn a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.

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 α2-antiplasmin. Microplasmin chimerization had minimal (∼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.

Fibrinolysis is amplified by converting α2‐antiplasmin from a plasmin inhibitor to a substrate

Summary.  α2‐antiplasmin (α2‐AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against α2‐AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of α2‐AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to α2‐AP with 5‐fold to 10‐fold higher relative affinity than mAb‐RWR, while mAb‐RWR bound with greater avidity to immobilized or denatured α2‐AP. Binding studies with chimeric α2‐APs revealed that none of the mAbs bound to sites in α2‐AP that form putative contacts with plasmin, namely the carboxy terminal lysines of α2‐AP, or the reactive center loop in the serpin domain of α2‐AP. Rather, mAb‐RWR recognized an epitope in the amino‐terminus of α2‐AP (L13GNQEPGGQTALKSPPGVCS32) near the site at which α2‐AP cross‐links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of α2‐AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by α2‐AP (from 1.1 ± 0.1 to 51 ± 4 and 67 ± 7) indicating that they convert α2‐AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of α2‐AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known α2‐AP–plasmin contacts to enhance fibrinolysis by converting α2‐AP from an inhibitor to a plasmin substrate.

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator

Summary.  Background: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA’s fibrin‐targeted properties that focus plasminogen activation on the fibrin surface. Objective: We examined whether re‐programming SK’s mechanism of action would create PAs with greater fibrin‐targeting and potency than TPA. Methods and Results: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKΔ1 and SKΔ59 were 5‐fold and > 119‐fold more fibrin‐dependent than SK (P < 0.0001), and 2‐fold and > 50‐fold more fibrin‐dependent than TPA (P < 0.001). The marked fibrin‐targeting of SKΔ59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by α2‐antiplasmin, and (iii) it only processed fibrin‐bound plasminogen. To assess the fibrin‐targeting and therapeutic potential of these PAs in vivo, a novel ‘humanized’ fibrinolysis model was created by reconstituting plasminogen‐deficient mice with human plasminogen. When compared with TPA, SKΔ1 and SKΔ59 were 4‐fold (P < 0.0001) and 2‐fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass‐dose basis and 2–3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. Conclusion: These experiments suggest that reprogramming SK’s mechanism of action markedly enhances fibrin‐targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

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.

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.