Presence Of rt-PA Research Articles

Effect of Andexanet-TFPI Interaction on in Vitro Thrombin Formation and Coagulation Markers in the TF-Pathway

Background: Tissue factor pathway inhibitor (TFPI) plays a major role in regulation of tissue factor (TF)-initiated coagulation in a FXa-dependent manner. Andexanet alfa (AnXa) is a modified, recombinant, catalytically inactive form of human FXa being developed to reverse the anticoagulant activity of FXa inhibitors in patients during episodes of major bleeding. As a modified FXa, AnXa retained high binding affinity to fXa inhibitors, including TFPI. Our previous in vitro studies demonstrated that AnXa reverses rivaroxaban-induced inhibition of thrombin generation (TG) initiated by either the extrinsic or intrinsic pathways. However, the thromboelastography (TEG) results showed that AnXa-TFPI interaction enhanced thrombin formation at low TF conditions, which suggested potential increase in the fibrin degradation product in the presence of recombinant tissue plasminogen activator (rtPA) (Lu, G., et al, ASH2016). In the current study, we further characterized the differential effect of AnXa on thrombin formation and clot lysis via the two different pathways, and correlated the TG and TEG parameters with the coagulation markers in the TF-initiated reaction in plasma, including prothrombin fragment (F1+2), thrombin-antithrombin complex (TAT), and fibrin degradation product (D-dimer).Methods: TF-initiated TG in human plasma was measured using a calibrated automated thrombogram (TF-CAT) and the PPP-reagent (5 pM TF, Diagnostica Stago). Non-TF-initiated TG was measured using CAT and an aPTT reagent, Actin FS (Siemens Healthcare) as the activator (Actin FS-CAT). Clot formation was measured in plasma using a TEG 5000 analyzer (Haemonetics). TF (1 pM) and Actin FS (1:240 final dilution) were used as the activators with or without rtPA (150 ng/mL). Time-dependent thrombin formation and clot lysis were monitored by quenching samples at various times (0, 2.5, 5, 10, 15, 30, 60 min) following initiation of the reaction by TF (1 pM) in plasma with or without rtPA (150 ng/mL) and AnXa (4.0 µM). Corn trypsin inhibitor (CTI, 50 µg/mL) was added to block contact activation. F1+2, TAT and D-dimer were quantified according to manufacturers' recommendations.Results: The effect of AnXa on TF-CAT and Actin FS-CAT were compared side-by-side with AnXa at low (0 - 20 nM) and high (0 - 4.0 µM) concentration ranges. AnXa (5 nM) increased TF-CAT parameters sensitive to TFPI activity (i.e., peak thrombin), with less effect on endogenous thrombin potential (ETP). No further increase in these parameters was observed at AnXa therapeutic concentrations (e.g., 2.0 and 4.0 µM), consistent with the expected effect of AnXa on the activity of TFPI, which is present at low plasma concentration (~2.4 nM). In contrast, AnXa did not significantly affect the Actin FS-CAT parameters.The effect of AnXa-TFPI interaction on coagulation and fibrinolytic pathways in human plasma were compared using TEG, with or without rtPA. AnXa (4.0 µM) reduced the TF-TEG-R parameter (lag time, equivalent to clotting time) and resulted in an increased area under the fibrinolytic profile with rtPA as previously shown. In contrast, AnXa and the buffer controls had similar profiles in the Actin FS-TEG assay under similar conditions, with or without rtPA (150 ng/mL) in the current studies, suggesting lack of an AnXa-TFPI effect.We further investigated the effect of AnXa-TFPI interaction on coagulation markers following TF (1pM)-initiated thrombin formation in plasma. AnXa (4 µM) increased F1+2 and TAT levels at early time points following initiation (2.5, 5, 10, 15 min), but had no difference at or after 30 min, compared to buffer control. A similar pattern was observed for D-dimer level when exogenous rtPA (150 ng/mL) was added. Compared to buffer control, AnXa caused elevation of D-dimer level at early time points but had no difference at or after 30 min following initiation of the reaction.Conclusions: AnXa-TFPI interaction can modulate the TF/TFPI activity in the initiation phase of the extrinsic coagulation pathway and increase parameters sensitive to TFPI activity in the TF-CAT and TF-TEG assays, but has minimal effect on the later phase of the reactions and the overall thrombin formation potential. Consistent with clinical observations, AnXa-TFPI interaction increased the F1+2 and TAT levels, as well as D-dimer level in the presence of rtPA in the early phase of the reaction following initiation of the reaction by TF. DisclosuresLu:Portola Pharmaceuticals, Inc.: Employment. Lin:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

Andexanet Alfa, a Universal Antidote Under Development for Factor Xa Inhibitors, Reverses Rivaroxaban-Induced Inhibition of Thrombin Generation Initiated By the Intrinsic Coagulation Pathway Independent of TFPI

Background: Andexanet alfa (AnXa) is a modified, recombinant human fXa molecule being developed to reverse the anticoagulant activity of fXa inhibitors in patients during episodes of major bleeding. As a modified fXa, AnXa retained high binding affinity to fXa inhibitors and had no significant interaction with major plasma coagulation proteins, except for tissue factor pathway inhibitor (TFPI), an endogenous fXa inhibitor with sub-nanomolar affinity to both fXa and AnXa. In previous in vitro and clinical studies in healthy volunteers, AnXa has shown dose-dependent and complete reversal of both direct and indirect fXa inhibitors in tissue factor (TF)-initiated thrombin generation (TG). To delineate the contribution of AnXa-TFPI interactions to TG, we compared rivaroxaban (Riva)-induced inhibition of TG initiated via the extrinsic pathway (TF) versus the intrinsic pathway (non-TF). The differential effect of AnXa on TG and clot formation via the two pathways was further studied in the context of recombinant tissue plasminogen activator (rtPA)-induced fibrinolysis using thromboelastography (TEG).Methods: TF-initiated TG in human plasma was measured using a calibrated automated thrombogram (CAT) and the PPP-reagent (5 pM TF). Non-TF-initiated TG was measured using CAT and Actin FS, an aPTT reagent. Anti-fXa activity was measured using a modified anti-fXa chromogenic assay with reagents from the Coamatic Heparin kit (DiaPharma). Riva was used as the standard. Pooled human plasma was spiked with Riva (0 - 2 µM) or Riva (1 µM)+AnXa (0 - 4 µM), and TG and anti-fXa activity were measured. Clot formation with or without AnXa was measured in plasma using a TEG 5000 analyzer. The functional fibrinogen (TF) and Kaolin reagents (both from Haemonetics) were used according to the manufacturer’s instruction. For low TF-initiated clot formation, the PPP-reagent (5 pM TF) was diluted in phospholipid (4.0 µM) followed by measuring the TEG profiles with 0.25, 0.5 and 1.0 pM TF.Results: The potential contribution of AnXa-TFPI interaction to TG was studied under similar conditions using TF or Actin FS in human plasma containing AnXa, Riva or Riva+AnXa. AnXa alone had minimal effect on the endogenous thrombin potential (ETP) in either assay. AnXa was able to fully reverse Riva-induced anticoagulation in the Actin FS TG assay, independent of AnXa-TFPI interaction that primarily modulates TF activity. The modulation of TF activity could be assessed by correlating ETP vs. the anti-fXa activity in samples containing Riva or Riva+AnXa. Riva dose-dependently inhibited TF-initiated TG as anti-fXa activity increased. At similar anti-fXa levels (i.e., similar free Riva concentration), Riva+AnXa had higher ETP than Riva alone. As expected, this difference was not observed in the Actin FS TG assay.To further investigate the role of AnXa-TFPI interaction on coagulation and fibrinolytic pathways, the profile of clot formation was studied in human plasma using TEG without Riva. AnXa (4 µM) had no effect on the TEG parameters in the kaolin assay or the functional fibrinogen assay containing high TF, with or without rtPA (150 ng/mL). When low TF (0.25, 0.5, and 1.0 pM) was used to initiate clot formation in the absence of rtPA, AnXa reduced the TEG-R parameter (lag time equivalent to clotting time), but had no effect on maximum amplitude (MA). The fibrin clot formed under each condition (±AnXa) was lysed slowly at low rtPA (75 ng/mL, ~1 nM), resulting in well-segregated processes of coagulation and fibrinolysis. However, rtPA at 150 ng/mL (~2 nM) dramatically changed the lysis profiles that overlapped the fibrin formation, possibly due to the higher rtPA activity ratio, relative to the major plasma inhibitor PAI-1 (<1 nM). With the optimal rtPA, fibrin clot formed at each TF concentration (±AnXa) was compensated by the fibrinolytic activity of rtPA resulting in an increased fibrin degradation product.Conclusions: In the absence of a fXa inhibitor, AnXa had minimal effect on TF or Actin FS-initiated TG with no direct interaction or effect on rtPA function. AnXa dose-dependently and completely reversed Riva-induced inhibition of TG initiated by either the intrinsic or extrinsic pathway, but had different effect on ETP due to the AnXa-TFPI interaction that mainly modulates TF function. AnXa-TFPI interaction may enhance TF-initiated TG at low TF conditions with increased fibrin degradation product in the presence of rtPA. DisclosuresLu:Portola Pharmaceuticals, Inc.: Employment. Lin:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Conley:Portola Pharmaceuticals, Inc.: Employment.

Efficacy of histotripsy combined with rt-PA in vitro

Histotripsy, a form of therapeutic ultrasound that uses the mechanical action of microbubble clouds for tissue ablation, is under development to treat chronic deep vein thrombosis (DVT). We hypothesize that combining thrombolytic agents with histotripsy will enhance clot lysis. Recombinant tissue plasminogen activator (rt-PA) and rt-PA-loaded echogenic liposomes that entrain octafluoropropane microbubbles (OFP t-ELIP) were used in combination with highly shocked histotripsy pulses. Fully retracted porcine venous clots, with similar features of DVT occlusions, were exposed either to histotripsy pulses alone (peak negative pressures of 7–20 MPa), histotripsy and OFP t-ELIP, or histotripsy and rt-PA. Microbubble cloud activity was monitored with passive cavitation imaging during histotripsy exposure. The power levels of cavitation emissions from within the clot were not statistically different between treatment types, likely due to the near instantaneous rupture and destruction of OFP t-ELIP. The thrombolytic efficacy was significantly improved in the presence of rt-PA. These results suggest the combination of histotripsy and rt-PA could serve as a potent therapeutic strategy for the treatment of DVT.

Abstract WP73: No Influence of Dabigatran or its Specific Reversal Agent, Idarucizumab, on rTPA-induced Thrombolysis of Clots in Human Plasma: an in vitro Study

Oral anticoagulation reduces the elevated incidence of stroke in patients with atrial fibrillation. Though stroke risk is reduced, it is not entirely prevented. When patients present with ischemic stroke and are also being treated with newer oral anticoagulants, such as dabigatran, this can complicate therapy. Currently a specific reversal agent to dabigatran, idarucizumab, is in development and was shown to immediately reverse the anticoagulant effect of dabigatran in volunteers and patients. However, it is unknown if administration of idarucizumab to reverse dabigatran results in any nonspecific effects on the action of rtPA. Thus this study was performed to determine the effect of dabigatran and its reversal with idarucizumab on rtPA-induced thrombolysis of a clot in human plasma in vitro. Blood was obtained from volunteers after written informed consent. Clots were produced by adding thromboplastin and 200mM CaCl2 to platelet rich plasma containing 594Alexa-labelled fibrinogen at 37°C for 60 min. Clots were then removed, washed and added to plasma from the same subject containing 150 IU/mL rtPA ± dabigatran (150 ng/mL) for 1 hr. Idarucizumab (3 mg/mL) was added after 60 min and incubated for a further 60 min at 37°C, all under continuous stirring. Lysis was measured as fluorescence released into plasma from the clot. Data shown as mean ± SD, n=5, were compared with ANOVA, p&lt;0.05 was significant. Initial clot weight was similar (18±6 mg) across groups. Clots added to plasma containing only dabigatran or idarucizumab underwent no lysis. Incubation with rtPA resulted in 28±17, 34±18 and 61±18% lysis after 60, 65 and 120 min, respectively (p&lt;0.001 vs control). Addition of dabigatran or idarucizumab alone did not significantly affect lysis in the presence of rtPA. Addition of dabigatran (at t=0) followed by idarucizumab (at t=60 min) to plasma resulted in similar rtPA-induced thrombolysis, 30±16, 34±14 and 55±17%, as compared to incubation with rtPA alone. Clot weight after 2 hrs was 6.3±2.2 mg with no difference across groups. These data show there are no non-specific interactions of idarucizumab with rtPA-induced thrombolysis in this in vitro model. It reinforces the specificity of idarucizumab for only dabigatran and its lack of binding to other substrates.

Clot Retraction Affects the Extent of Ultrasound-Enhanced Thrombolysis in an Ex Vivo Porcine Thrombosis Model

We investigated ultrasound-enhanced thrombolysis in two whole-blood clot models using a Food and Drug Administration-approved contrast agent (Definity, Lantheus Medical Imaging; Billerica, MA USA) and thrombolytic drug (recombinant tissue-type plasminogen activator [rt-PA]) (Genentech; South San Francisco, CA USA). Porcine venous blood was collected from donor hogs and coagulated in vials made of two different materials. This method produced clots with differing compositional properties, as determined by routine scanning electron microscopy and histology. Clots were deployed in an ex vivo porcine thrombosis model, and exposed to an intermittent ultrasound scheme previously developed to maximize stable cavitation while acoustic emissions were detected. Exposure to 3.15 μg/mL rt-PA promoted lysis in both clot models, compared with exposure to plasma alone. However, only unretracted clots experienced significant enhancement of thrombolysis in the presence of rt-PA, Definity, and ultrasound, compared with treatment with rt-PA. In these clots, microscopy revealed loose erythrocyte aggregates, a significantly less extensive fibrin network and a higher porosity, which may facilitate increased penetration of thrombolytics by cavitation.

Memantine Improves Safety of Thrombolysis for Stroke

Despite side effects including N-methyl-d-aspartate-mediated neurotoxicity, recombinant tissue-type plasminogen activator (rtPA) remains the only approved acute treatment for ischemic stroke. Memantine, used for treatment of Alzheimer disease, is an antagonist for N-methyl-d-aspartate receptors. We investigated whether memantine could be used as a neuroprotective adjunct therapy for rtPA-induced thrombolysis after stroke. In vitro N-methyl-d-aspartate exposure, oxygen and glucose deprivation, and N-methyl-d-aspartate-mediated calcium videomicroscopy experiments were performed on murine cortical neurons in the presence of rtPA and memantine. The therapeutic safety of rtPA and memantine coadministration was evaluated in mouse models of thrombotic stroke and intracerebral hemorrhage. Ischemic and hemorrhagic volumes were assessed by MRI and neurological evaluation was performed by the string test and automated gait analysis. Our in vitro observations showed that memantine was able to prevent the proneurotoxic effects of rtPA in cultured cortical neurons. Although memantine did not alter the fibrinolytic activity of rtPA, our in vivo observations revealed that it blunted the noxious effects of delayed thrombolysis on lesion volumes and neurological deficits after ischemic stroke. In addition, memantine rescued rtPA-induced decrease in survival rate after intracerebral hemorrhage. Memantine could be used as an adjunct therapy to improve the safety of thrombolysis.

Compatibility of Recombinant Tissue Plasminogen Activator and Bevacizumab Co-Applied for Neovascular Age-Related Macular Degeneration With Submacular Hemorrhage

To investigate the compatibility of recombinant tissue plasminogen activator (rtPA) and bevacizumab in vitro because during surgery, rtPA or rtPA-induced plasmin may cleave and inactivate bevacizumab. To simulate the intraoperative range of mixing ratios of rtPA, bevacizumab, and subretinal blood, we calculated the volumes of 12 submacular hemorrhages (SHs) with a spherical cap formula using measurements derived from fundus photographs and spectral-domain optical coherence tomographic images. Bevacizumab was incubated with rtPA or plasmin before gel electrophoresis with Coomassie blue and silver staining. The anti-angiogenetic activity of bevacizumab in the presence of rtPA with or without clotted human blood or of plasmin was quantified by vascular endothelial growth factor–enzyme-linked immunosorbent assay after incubation with the supernatant of porcine retinal pigment epithelium cell cultures. The mean (SD) volume of SH was 28.6 (24.7) mm3 (range, 6.2-94.6 mm3). In sodium dodecyl sulfate–polyacrylamid electrophoresis with Coomassie blue or silver staining, bevacizumab displayed characteristic patterns of protein bands. No additional fragments were detected in co-application of bevacizumab with either rtPA or plasmin. The anti-angiogenetic activity of bevacizumab remained unchanged in co-application with rtPA with or without blood or plasmin. We demonstrated the absence of cleavage or functional inactivation of bevacizumab by rtPA in an in-vitro model of their intraoperative co-application as a treatment of SH. In clinical practice, rtPA and bevacizumab can be co-applied as a treatment for neovascular age-related macular degeneration with SH to simultaneously clear SH and reduce choroidal new vessel activity.

Short-term High-Dose Effect of Lovastatin on Thrombolysis by rt-PA in a Human Whole-Blood in vitro Clot Model

High-dose hydroxymethylglutaryl coenzyme. A reductase inhibitor (statin) administration reduces neuronal injury and improves outcomes in experimental models of acute ischemic stroke, and has been shown to be safe in a phase 1 dose-escalation study using lovastatin at doses higher than currently approved for daily use. Statins also affect the hemostatic system by upregulating t-PA expression and decreasing plasminogen activator inhibitor (PAI-1) expression, platelet adhesion and thrombus formation in animal models. Since a thrombolytic agent, recombinant tissue plasminogen activator (rt-PA), is currently the only FDA-approved therapy for use in ischemic stroke patients, it is important to ascertain whether high statin doses impact the efficacy of rt-PA. The main goal of this study was to evaluate the effect of a high dose of lovastatin and its active form, lovastatin hydroxy acid, on rt-PA thrombolysis in an in vitro model. Percentage clot lysis was measured in the presence and absence of rt-PA in three different treatment groups: lovastatin, lovastatin hydroxy acid, and ethanol. The effect of ethanol on clot lysis was studied since ethanol was used to disperse the highly hydrophobic lovastatin. The decrease in clot width over time was measured using microscopic imaging of an in vitro human whole blood clot model; an approximately 400 μm diameter clot was formed on suture silk, suspended in human fresh frozen plasma (hFFP) and exposed to treatment. In the absence of rt-PA, clot lysis did not show statistically significant differences in the percentage clot lysis between different treatment groups (p=0.103). In the presence of rt-PA, clot lysis was greater than in the absence of rt-PA for all groups, but there were no statistically significant differences between treatment groups (p=0.385). In this in vitro study, high doses of lovastatin neither impaired nor enhanced the lytic efficacy of rt-PA.

The JNK Inhibitor XG-102 Protects from Ischemic Damage with Delayed Intravenous Administration Also in the Presence of Recombinant Tissue Plasminogen Activator

Background: XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide which selectively inhibits the c-Jun N-terminal kinase, is a powerful neuroprotectant in mouse models of middle cerebral artery occlusion (MCAo) with delayed intracerebroventricular injection. We aimed to determine whether this neuroprotection could also be achieved by intravenous injection of XG-102, which is a more feasible approach for future use in stroke patients. We also tested the compatibility of the compound with recombinant tissue plasminogen activator (rtPA), commonly used for intravenous thrombolysis and known to enhance excitotoxicity. Methods: Male ICR-CD1 mice were subjected to a 30-min-suture MCAo. XG-102 was injected intravenously in a single dose, 6 h after ischemia. Hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mM) deprivation for 30 min. rtPA was added after ischemia and before XG-102 administration, both in vitro and in vivo. Results: The lowest intravenous dose achieving neuroprotection was 0.0003 mg/kg, which reduced the infarct volume after 48 h from 62 ± 19 mm³ (n = 18) for the vehicle-treated group to 18 ± 9 mm³ (n = 5, p < 0.01). The behavioral outcome was also significantly improved at two doses. Addition of rtPA after ischemia enhanced the ischemic damage both in vitro and in vivo, but XG-102 was still able to induce a significant neuroprotection. Conclusions: A single intravenous administration of XG-102 several hours after ischemia induces a powerful neuroprotection. XG-102 protects from ischemic damage in the presence of rtPA. The feasibility of systemic administration of this promising compound and its compatibility with rtPA are important steps for its development as a drug candidate in ischemic stroke.

The use of the Chandler loop to examine the interaction potential of NXY‐059 on the thrombolytic properties of rtPA on human thrombi in vitro

Recombinant tissue-type plasminogen activator (rtPA) is the only globally approved treatment for acute ischaemic stroke. Other potential treatments might be administered with rtPA, making it important to discover whether compounds interfere with rtPA-induced lysis. We evaluated methods for examining the effect of the neuroprotectant NXY-059 on the lytic property of rtPA. Plasma clot formation and lysis in the presence of rtPA and NXY-059 was measured as the change in plasma turbidity. The effect of NXY-059 on rtPA-induced lysis was similarly assessed on preformed clots. Lysis of the thrombus formed in a Chandler loop measured release of fluorescent-tagged fibrinogen that had been incorporated during thrombus formation. Thrombi were exposed to both rtPA and NXY-059 throughout lysis in the presence of 80% autologous plasma and the release of label during lysis was measured. Data interpretation is limited in the clot lysis experiments because either the rtPA was present during clot formation or the drug was added to a clot formed in static conditions. In contrast, thrombi were formed in dynamic flow conditions in the Chandler loop and the time course of lysis in plasma was examined. rtPA increased thrombolysis and the antifibrinolytic trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCA) inhibited lysis. Lysis induced by rtPA was unaltered by NXY-059. The Chandler loop method provides a reliable technique for examining the effect of compounds on rtPA-induced lysis in vitro and demonstrated that NXY-059 does not alter rtPA-induced lysis at clinically relevant concentrations of either drug.

The Effect of Anticonvulsant Drugs on the Fibrinolytic Activity of Tissue Plasminogen Activator

Background and Purpose: Several anticonvulsant drugs have been found to be neuroprotective in preclinical models of stroke, and such drugs may possibly be given in combination with other stroke treatments such as recombinant tissue plasminogen activator (rt-PA). The goal of this study was to test for potential interactions between rt-PA and selected anticonvulsants. Methods: A spectrophotomeric assay was used to monitor the lysis of fibrin clots in the presence of rt-PA and the drugs levetiracetam, valproic acid, phenytoin and phenobarbital. Results: The drugs tested were found to have no effect on either the rate or total amount of lysis induced by rt-PA. Conclusions: Although further studies are required in order to explore the effects of these drugs in stroke patients, the results suggest that co-administration of rt-PA and anticonvulsant drugs may be safe and viable.

Ultrasound-enhanced tissue plasminogen activator thrombolysis in an in vitro porcine clot model

IntroductionThrombolytics such as recombinant tissue plasminogen activator (rt-PA) have advanced the treatment of ischemic stroke, myocardial infarction, deep vein thrombosis and pulmonary embolism. ObjectiveTo improve the efficacy of this thrombolytic therapy, the synergistic effect of rt-PA and 120 kHz or 1.0 MHz ultrasound was assessed in vitro using a porcine clot model. Materials and methodsFully retracted whole blood clots prepared from fresh porcine blood were employed to compare rt-PA thrombolytic treatment with and without exposure to 120-kHz or 1-MHz ultrasound. For sham studies (without ultrasound), clot mass loss was measured as a function of rt-PA concentration from 0.003 to 0.107 mg/ml. For combined ultrasound and rt-PA treatments, peak-to-peak pressure amplitudes of 0.35, 0.70 or 1.0 MPa were employed. The range of duty cycles varied from 10% to 100% (continuous wave) and the pulse repetition frequency was fixed at 1.7 KHz. ResultsFor rt-PA alone, the mass loss increased monotonically as a function of rt-PA concentration up to approximately 0.050 mg/ml. With ultrasound and rt-PA exposure, clot mass loss increased by as much as 104% over rt-PA alone. Ultrasound without the presence of rt-PA did not significantly enhance thrombolysis compared to control treatment. The ultrasound-mediated clot mass loss enhancement increased with the square root of the overall treatment duration. ConclusionsBoth 120-kHz and 1-MHz pulsed and CW ultrasound enhanced rt-PA thrombolysis in a porcine whole blood clot model in vitro. No clear dependence of the observed thrombolytic enhancement on ultrasound duty cycle was evident. The lack of duty cycle dependence suggests a more complex mechanism that could not be sustained by merely increasing the pulse duration.

Correlation of cavitation with ultrasound enhancement of thrombolysis

Pulsed ultrasound, when used as an adjuvant to recombinant tissue plasminogen activator (rt-PA), has been shown to enhance thrombolysis in the laboratory as well as in clinical trials for the treatment of ischemic stroke. The exact mechanism of this enhancement has not yet been elucidated. In this work, stable and inertial cavitation (SC and IC) are investigated as possible mechanisms for this enhancement. A passive cavitation detection scheme was utilized to measure cavitation thresholds at 120 kHz (80% duty cycle, 1667 Hz pulse repetition frequency) for four host fluid and sample combinations: plasma, plasma with rt-PA, plasma with clot and plasma with clot and rt-PA. Following cavitation threshold determination, clots were exposed to pulsed ultrasound for 30 min in vitro using three separate ultrasound treatment regimes: (1) no cavitation (0.15 MPa), (2) SC alone (0.24 MPa) or (3) SC + IC combined (0.36 MPa) in the presence of rt-PA. Percent clot mass loss after each treatment was used to determine thrombolysis efficacy. The highest percent mass loss was observed in the stable cavitation regime (26%), followed by the combined stable and inertial cavitation regime (20.7%). Interestingly, the percent mass loss in clots exposed to ultrasound without cavitation (13.7%) was not statistically significantly different from rt-PA alone (13%) [ p > 0.05]. Significant enhancement of thrombolysis correlates with presence of cavitation and stable cavitation appears to play a more important role in the enhancement of thrombolysis. (E-mail: saurabhdattas@yahoo.com)

Differential Effects of c7E3 Fab on Thrombus Formation and rt-PA-Mediated Thrombolysis Under Flow Conditions

Although the Fab fragment of the mouse–human chimeric anti-α IIbβ 3 (GP IIb/IIIa) monoclonal antibody (MoAb) c7E3 facilitates recombinant tissue-type plasminogen activator (rt-PA)-mediated thrombolysis, it is not clear whether this is due to inhibition of new clot formation and/or a direct effect on the lysis rate. We employed an in vitro flow (re)circulation model to investigate how c7E3 Fab affected (a) platelet adhesion to clotted fibrin substrates under laminar flow at wall shear rates of 100 or 500 s −1 and (b) rt-PA-induced lysis of preformed mural platelet–fibrin substrates at 500 s −1. c7E3 Fab dose-dependently (0.5–5 μg/ml) inhibited platelet adhesion from flowing whole blood onto fibrin substrates (∼14 μm thick) at each wall shear rate. When at 5 min after the onset of flow, c7E3 Fab (0.1–10 μg/ml) and rt-PA (1 μg/ml) were coinjected in flowing blood, it was found that modest fibrinolysis caused major platelet release from fibrin substrates and there was no difference in the lysis rate in the presence of rt-PA+c7E3 Fab compared to rt-PA alone. Platelet pretreatment with c7E3 Fab (10 μg/ml) had no effect on the lysis rate of thin (∼40 μm), and slightly delayed the lysis rate of thick (<250 μm), platelet–fibrin substrates containing evenly dispersed platelets (10 9/ml). When the platelets within thick platelet–fibrin substrates were organized in platelet-rich regions (“residual thrombi”), these substrates followed a nonuniform lysis pattern, where fibrin between the thrombi lysed first and the residual thrombi lysed at a slower rate. Platelet pretreatment with c7E3 Fab (10 μg/ml) abolished the formation of the lytic-resistant residual thrombi and the associated platelet-protected fibrin zones. Hence, treatment with c7E3 Fab has no direct effect on the rate of rt-PA-mediated lysis, but is expected to block platelet–fibrin interactions that lead to clot retraction, thus maintaining a fibrin architecture that is more susceptible to lysis.

Membrane lipid fluidity of blood platelets: a common denominator that underlies the opposing actions of various agents that affect platelet activation in whole blood

Membrane lipid fluidity (MLF) is thought to play a crucial role in signal transduction and is believed to affect the responsiveness of blood platelets. In a recent study it was demonstrated that EDTA, used as the blood anticoagulant, brought about a significant increase in expression of GMP-140 antigen, and this effect was accompanied by a significant increase in platelet MLF. Moreover, this spontaneous EDTA-driven platelet activation was vastly attenuated in the presence of tissue-type plasminogen activator, which is also known to affect platelet MLF. The hypothesis was raised that the modulation of platelet membrane fluidity by EDTA might underlie platelet spontaneous activation in the presence of EDTA. To further explore the possible molecular mechanism(s) of the EDTA-dependent triggering of signal transduction pathway(s) in human blood platelets, we monitored the extent of spontaneous platelet activation in the presence of EDTA and selected platelet membrane 'fluidizers' and 'rigidizers'. A reduction in the EDTA-dependent platelet release and activation was noted, not only in the presence of rt-PA (by over 50%, P < 0.001), which acted as a rigidizer of platelet membrane fluidity (ESR h+1/h0 ratios of 5-DOXYL-Ste and 12-DOXYL-Ste decreased by 6.2%, P 0.0001, and 3.8%, P < 0.02, respectively), but also in the presence of other modulators of MLF, regardless of their fluidizing or rigidizing effects. Both rigidizers (procaine and lidocaine, 5-DOXYL-Ste h+1/h0 reduced by up to 6.5%, 12-DOXYL-Ste h+1/h0- by up to 4.5%, P < 0.02 or less) and fluidizers (benzyl alcohol, ethanol, 12-DOXYL-Ste h+1/h0 increased by 17.8% and 6.1%, respectively, P 0.0001) of platelet membranes significantly depressed platelet activation (respectively, down to 1.1%, 7.7%, 6.7% and 8.5% vs control EDTA 22.9% of CD62-positive platelets). We suggest that EDTA induces alterations in membrane glycoprotein structure and affect MLF by altering lipid-protein interactions, and thus triggers signal transduction in the course of platelet activation. The resulting displacements in platelet membrane proteins, dislocation of membrane components and/or distortion of lipid-protein interactions could generate an 'outside-in' signalling that is mediated by the altered platelet MLF. Overall, it is likely that interference with the structure and conformation of selected domains of platelet membrane proteins might be the crucial mechanism by which EDTA leads to exaggerated activation of platelets in whole blood.

IMPAIRED CLOT LYSIS BY rt-PA CATALYZED MINI-PLASMINOGEN ACTIVATION

The fibrinolytic system contains a proenzyme plasminogen (Plg) which is converted to plasmin (Plm) by the action of Plg activators. Physiological Plg activators are: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plg was shown to be further cleaved by leukocyte elastase producing several fragments, one of wich is called mini-plasminogen (mini-Plg) or neo-plasminogen Val 442. In this paper we studied whether mini-Plg is able to produce clot lysis when it is activated by rt-PA in purified systems and in Plg depleted normal plasma. We found that mini-Plg clot lysis time was longer than that of Plg. Clot lysis times were 2.3 minutes ± 0.06 for Plg and 9.8 minutes ± 0.1 for mini-Plg. Mini-Plg is less efficient than Plg in producing clot lysis at all studied concentrations (0.1–1.2μM). In Plg depleted normal human plasma mini-Plg is unable to produce complete clot lysis in presence of rt-PA. Although mini-Plg can be activated to mini-Plm by rt-PA, these results show that the activation process is insufficient to produce an efficient clot lysis. © 1997 Elsevier Science Ltd

Improved Mechanical Thrombolysis with the Addition of Recombinant Tissue-Type Plasminogen Activator: In Vitro Study with Use of Cr-51–labeled Clots

To explore the potential effect of infusion of recombinant tissue-type plasminogen activator (rt-PA) during mechanical thrombolysis. Clots containing chromium-51-labeled platelets produced from blood from two donors were placed in a solution of rt-PA (1.25 micrograms/mL) or in saline solution and were pulverized with a mechanical thrombolysis catheter. The resultant slurry was passed through a filter with a pore size of 10 microns, and the radioactivity of the clot residue on the filter was measured. For each donor, the percentage of clot remaining was calculated from the residual radioactivity. The mean percentages of clot remaining were 72.0% for clots in saline (n = 10) and 44.4% for clots in rt-PA (n = 10) in donor 1 and 58.9% (n = 14) and 36.4% (n = 14), respectively, for donor 2. In both cases, the mean percentage of clot remaining was significantly lower when mechanical thrombolysis was performed in the presence of rt-PA. These results indicate the feasibility of combined pharmacologic and mechanical thrombolysis.

Synthesis and secretion of plasminogen activator inhibitor 1 by human endothelial cells in vitro. Effect of active site mutagenized tissue-type plasminogen activator.

The effects of recombinant tissue-type plasminogen activator (rt-PA) and of an inactive mutant of rt-PA, obtained by mutagenesis of the active site Ser478 to Ala (rt-PA-Ala478), on the synthesis and secretion of plasminogen activator inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) in culture were studied. Under base-line conditions, PAI-1 antigen secretion was 4.3 +/- 1.0 micrograms (mean +/- S.D., n = 8) per 10(6) cells in 24 h. This PAI-1 had a low specific activity (6,000 +/- 1,600 units/mg) and Mr of 50,000, which was not altered by addition of rt-PA. In HUVEC cultured with 2 micrograms/ml rt-PA-Ala478, PAI-1 antigen secretion was 2.1 +/- 0.8 micrograms (n = 5) per 10(6) cells in 24 h with a specific activity of 120,000 +/- 42,000 units/mg and Mr of 50,000. Addition of rt-PA to this conditioned medium resulted in generation of three main components: 16% migrated as an Mr 106,000 rt-PA.PAI-1 complex, 16% as an Mr 81,000 degraded rt-PA.PAI-1 complex and the remainder as an Mr 45,000 degradation product of PAI-1. HUVEC cultured with 2 micrograms/ml rt-PA secreted 3.9 +/- 0.6 micrograms (n = 8) PAI-1 antigen per 10(6) cells within 24 h, of which 20-50% occurred as intact or degraded complexes with t-PA (Mr 106,000 and 81,000) and the rest as an inactive Mr 45,000 degradation product of PAI-1. PAI-1 mRNA levels, determined by Northern blot analysis and expressed relative to beta-actin mRNA levels, were very similar for HUVEC cultured in the absence or the presence of rt-PA or rt-PA-Ala478. It is concluded that PAI-1 is secreted by HUVEC in culture in fully active form which spontaneously inactivates. PAI-1 can be stabilized by addition of rt-PA-Ala478 to the culture medium, resulting in a 20-fold increase in specific activity. Interaction of rt-PA with active PAI-1 produces both t-PA.PAI-1 complex and an inactive degradation product of PAI-1.