Direct-acting Thrombolytic Agent Research Articles

Cloning, purification and characterization of a recombinant protease with novel thrombolytic activity in human plasma and rat thrombosis models

BackgroundThere is a need to identify and develop novel thrombolytic agents that can directly digest fibrin clots from various biological resources. ObjectiveTo clone, express, purify, and characterize a recombinant protease named rvFMP capable of cleaving fibrinogen, fibrin polymer, and cross-linked fibrin in human plasma milieu and rat thrombosis model systems. ResultsWe cloned a vFMP-encoding gene from the genomic DNA of V. furnissii KCCM41679 using polymerase chain reaction (PCR), expressed in Escherichia coli, and purified rvFMP (stands for recombinant vibrio furnissii metalloprotease). The proteolytic activity of purified rvFMP enzyme could be clearly inhibited by 1,10-phenanthroline and ethylene glycol tetraacetic acid, but not by diisopropyl fluorophosphate, suggesting that it can be a typical metalloprotease. rvFMP showed an effective proteolytic activity in cleaving cross-linked fibrins in human plasma milieu. Remarkably, rvFMP exhibited a clear thrombolytic activity in rat thrombosis models such as ferric chloride-exposed rat carotid artery and carrageenan-treated rat tail. However, rvFMP (1.5 mg/kg) evoked no internal bleeding and also showed no lethal effect in mice. The recombinant enzyme also showed no cytotoxicity and had an inability to induce tumour necrosis factor-α (TNF-α) in Raw264.7 cells. ConclusionrvFMP can be a candidate enzyme capable of being developed as a novel direct-acting thrombolytic agent.

In vitro fibrinolysis and antithrombosis characterizations of novel recombinant microplasminogen with RGD and GPRP peptides.

Microplasminogen (μPlg), a truncated form of human plasminogen, has considerable potential as a direct-acting thrombolytic agent. To further develop μPlg into a thrombolytic agent with anti-thrombus properties, we constructed two μPlg variants containing tripeptide Arg-Gly-Asp (RGD) and tetrapeptide Gly-Pro-Arg-Pro (GPRP) by site-directed mutagenesis. The recombinant cDNAs were expressed in yeast (Pichia pastoris) and purified to high homogeneity by Ni-NTA affinity chromatography. The specific activities of RGD-μPlg and GPRP-μPlg were 7.7 and 13.3 U/mg, respectively, as determined using the fibrin-plate method. RGD-μPlg significantly inhibited ADP-induced platelet aggregation, which was 33.6- and 14.1-fold higher than the native μPlg and GPRP-μPlg, respectively. On the other hand, GPRP-μPlg prolonged thrombin-initialized fibrinogen polymerization in a concentration-dependent manner, which was 9.2- and 5.7-fold stronger than μPlg and RGD-μPlg, respectively. Under activation by urokinase, μPlg, RGD-μPlg, and GPRP-μPlg all showed over 80% conversions to their active enzyme in 24h. The structure models that docked RGD-μPlg and μPlg activation loops into the enzymatic active site of urokinase showed that Pro559 to Asp559 mutation of RGD-μPlg led to an alteration in the interaction, which possibly explains the slowed activation of RGD-μPlg by urokinase over an 80-min period. In conclusion, this study has presented two recombinant μPlg variants with anti-platelet aggregation and anti-fibrinogen clotting activity, thus suggesting the anti-thrombosis properties of these two μPlg derivatives.

Direct microscopic monitoring of initial and dynamic clot lysis using plasmin or rt-PA in an in vitro flow system

Plasmin is a direct-acting thrombolytic agent with a favorable safety profile upon intra-arterial delivery in pre-clinical and phase I studies. However, the thrombolytic efficacy of plasmin, relative to that of rt-PA, remains to be established. We have compared the dynamics of clot lysis with plasmin or rt-PA in an in vitro perfusion system, in which thrombolytic agent is administered locally, allowed to induce lysis for short intervals, then washed with plasma in a re-circulation circuit. Whole blood human clots were prepared in observation chambers, exposed to plasmin or rt-PA at equimolar concentrations (1.2/1.0, 1.8/1.5 and 2.4/2.0 mg/ml) for measured intervals of time, followed by perfusion with human plasma. Clot size was monitored by digital analysis of sequential photographs obtained through an optical microscope. Plasma perfusion after incubation with thrombolytic agent rapidly removed superficial clot fragments. This initial decrease in clot size was greater with plasmin than with rt-PA when tested at the highest concentrations of agent (0.63 ± 0.11 vs. 0.30 ± 0.11, p=0.001 for clots with non-cross-linked fibrin and 0.53 ± 0.15 vs. 0.14 ± 0.15, p=0.02, for clots with cross-linked-fibrin). Subsequent clot lysis during plasma flow was greater after prior incubation with rt-PA. Longer incubation times of plasmin resulted in larger portions of the clot being washed free. Repeated plasmin incubations and plasma perfusions of a clot successfully induced stepwise reductions in clot size. Initial clot lysis is greater with direct exposure using plasmin than rt-PA. During washout and circulation with plasma, rt-PA induced continued clot lysis, while plasmin lysis was curtailed, presumably because of plasmin inhibition.

Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke

BackgroundTissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat.MethodsMale spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm.ResultsThe main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2–3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats.ConclusionsThe superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.

Thrombolysis With Plasmin

Plasmin is a direct-acting thrombolytic agent with a striking hemostatic safety advantage over plasminogen activators in animal models of thrombolysis and bleeding. In contradistinction to plasminogen activators, which risk bleeding at any effective thrombolytic dose, plasmin is tolerated without bleeding at several-fold higher amounts than those needed for thrombolysis. Plasmin has been safe in a current trial in patients with peripheral arterial or graft occlusion, and efforts are now directed toward therapy of stroke caused by cerebral artery occlusion. A rabbit (4 kg body weight) model of 2-hour, thrombin-induced middle cerebral artery occlusion using angiographic documentation of vascular patency and recanalization was used to perform a dose-ranging study of plasmin, delivered by catheter over a median duration of 10 minutes. Plasmin induced early recanalization in all animals (3 per group) within 10 minutes after discontinuation of 3, 2, or 1 mg of agent infusion. Control saline infusion failed to induce recanalization in 3 of 3 subjects. Plasmin rapidly induces middle cerebral artery recanalization, as determined in an angiogram-based animal model of arterial occlusion. Based on these data and other information, a phase I/IIa clinical trial of plasmin in human middle cerebral artery ischemic stroke has been initiated.

Fibrolase: Trials and Tribulations

Fibrolase is the fibrinolytic enzyme isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria including reverse phase HPLC, molecular sieve chromatography and SDS-PAGE. The purified enzyme is a zinc metalloproteinase containing one mole of zinc. It is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. Fibrolase shares a significant degree of homology with enzymes of the reprolysin sub-family of metalloproteinases including an active site homology of close to 100%; it is rapidly inhibited by chelating agents such as EDTA, and by alpha2-macroglobulin (α2Μ). The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(β)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin, cleaving the α-chain more rapidly than the β-chain. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phaase II stroke trial, and Nuvelo dropped further development in 2008.

Safety and Efficacy of Alfimeprase in Subjects with Occluded Central Venous Access Devices: The SONOMA-2 Study.

Introduction: The infusion of chemotherapy and blood products is facilitated by central venous access devices (CVAD), and the occurrence of thrombotic CVAD occlusion results in delays in these crucial therapies. Alfimeprase is a direct-acting thrombolytic agent with the potential to rapidly re-establish catheter function.Methods: SONOMA-2 was a multicenter, multinational, randomized, double blind, placebo-controlled phase 3 study evaluating the efficacy and safety of alfimeprase in subjects with occluded CVADs. Subjects were randomized in a 2:1 ratio to receive either intra-luminal alfimeprase (A) at a dose of 3.0 mg or placebo (P). Study drug was delivered in a total volume of 2 mL. If re-establishment of catheter function was not established within 30 minutes, a second dose of the same study drug was instilled and allowed to dwell for an additional 30 minutes. Re-establishment of catheter function was assessed by an attempt to withdraw 3 mL of blood and infuse 5 mL of saline. The primary efficacy endpoint was re-establishment of a functional CVAD at 15 minutes post first infusion. The primary efficacy analysis prespecified a significance level of ≤ 0.00125.Results: A total of 303 patients (201 (A), 102 (P)) were randomized into the study. Treatment groups were well balanced at baseline with respect to overall demographics, catheter insertion site, and median days since last failed infusion or blood draw. The primary efficacy endpoint, re-establishment of a functional CVAD at 15 minutes post first infusion, was achieved in 34.3% (A) versus 21.6% (P) of subjects (p = 0.022). The cumulative proportion of subjects with re-establishment of a functional CVAD at 30 minutes post first or second infusions was 52.7% (A) versus 30.4% (P) (p = 0.0002). Rates of adverse events (65.8% (A), 59.4% (P)), serious adverse events (15.5% (A), 16.8% (P)), and hemorrhagic adverse events (3.6% (A), 10.9% (P)) were comparable between treatment groups. No intracranial hemorrhages were observed.Conclusions: Alfimeprase restored CVAD function in a numerically greater fraction of subjects than did placebo; however, the study did not achieve statistical significance for the primary endpoint at the prespecified p-value of ≤ 0.00125 necessary for demonstrating efficacy with a single pivotal study. Alfimeprase was well tolerated. Further clinical studies are underway that evaluate a higher and more concentrated alfimeprase dose which preclinical studies predict will result in greater efficacy. Alfimeprase remains a promising potential candidate for use in patients with CVAD occlusion.

A Phase I Trial of Alfimeprase for Peripheral Arterial Thrombolysis

To evaluate the safety profile, pharmacokinetics, and thrombolytic activity of alfimeprase, a novel direct-acting thrombolytic agent, in patients with chronic peripheral arterial occlusion (PAO). In this multicenter, open-label, single-dose, dose-escalation study, 20 patients with worsening symptoms of lower extremity ischemia within 6 months of enrollment were treated with alfimeprase in five escalating dose cohorts (0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.3 mg/kg, and 0.5 mg/kg) by means of intraarterial and sometimes intrathrombic pulsed infusion. The primary endpoint was safety assessed by adverse event rates. Additional safety assessments included vital sign monitoring, serum chemistry testing, hematologic testing, and coagulation testing for 28 days after the procedure, as well as alpha2-macroglobulin and antialfimeprase antibody testing for as long as 3 months after treatment. Pharmacokinetic parameters were evaluated with use of an assay that measures free and alpha2-macroglobulin-bound (ie, total) alfimeprase. No patient experienced a hemorrhagic adverse event. Mean plasminogen and fibrinogen concentrations were not substantially altered by treatment. Three transient treatment-related adverse events were reported in the same patient: one incidence each of increased blood fibrinogen level, skin rash, and headache. All three adverse events were graded as mild. The pharmacokinetic profile of alfimeprase suggested that the half-life for total alfimeprase ranges from 11 to 54 minutes (median, 25 min) in patients with PAO. The serum alpha2-macroglobulin concentrations decreased transiently in a dose response-like manner between 12 and 24 hours and returned to within normal limits approximately 14 days after alfimeprase exposure. Alfimeprase in doses as high as 0.5 mg/kg was generally well-tolerated in patients with chronic PAO. No bleeding complications were noted. The stable fibrinogen concentrations suggest that the activity of alfimeprase may be limited to the target thrombus. Alfimeprase holds the potential to achieve dissolution of thrombus with a diminished risk of hemorrhage.

Cross-Species Pharmacologic Evaluation of Plasmin as a Direct-Acting Thrombolytic Agent: Ex Vivo Evaluation for Large Animal Model Development

Human plasma-derived plasmin has been developed for the treatment of thrombosed hemodialysis arteriovenous grafts and vascular occlusive diseases. To further investigate this drug in large animal models and derive preliminary dosing estimates, the authors compared plasmin's relative lytic potential in four species, including man. The goal was to find which species' whole blood clots best compared to human clots in terms of lysis with plasmin. The results from these studies will serve to guide species selection for large animal experimentation. Clotted blood from human, pig, sheep, and bovine subjects were treated with saline solution control, plasmin, or tissue plasminogen activator. Electron microscopy (EM) techniques were used to investigate the effects of clot size and fragmentation on plasmin lysis, the effects of intrathrombic infusion by injection of plasmin directly into whole blood clots, and species fibrin structural differences. Under static conditions, plasmin efficiently lysed clots from all species studied at an optimal dose of 4-5 mg per 4-5 g of clot. With fragmented human clots, plasmin (5 mg)-induced lysis was 80% +/- 2% at 60 minutes. Porcine clots were more resistant to plasmin lysis compared with human, ovine, and bovine clots. Percent lysis at 60 minutes with plasmin for ovine clots was 72% +/- 3% (4-mg dose), compared with 50% +/- 4% for porcine clots (5-mg dose; P < .05). EM of porcine clots showed a compact fibrin network that appeared more dense than that in human or sheep clots, which may account for the decreased lytic rate. Human plasmin is an effective direct-acting thrombolytic agent that is capable of lysing fibrin from several species. Ex vivo lysis studies were used to investigate the most appropriate large animal model that best approximates plasmin lysis with human clots under certain conditions. It was determined that ovine clots treated with plasmin most closely resemble the lysis observed with human clots.

Supplemental Alpha-2-Antiplasmin Prevents Plasmin-Induced Hemorrhage.

Plasmin is a direct-acting thrombolytic agent with an impressive safety margin in experimental models, as it is tolerated without bleeding at a 6-fold greater dose than needed for thrombolysis. At higher plasmin dosages, above a reproducible dose threshold, alpha-2-antiplasmin (AP) is consumed, accompanied by complete depletion of fibrinogen and factor VIII. As a result of clotting factor exhaustion, the primary bleeding time is prolonged. We assessed whether supplemental AP could prevent bleeding induced by a purposefully toxic dosage of plasmin, and whether AP could arrest such bleeding once it occurs. Using the rabbit ear puncture model, plasmin (8 mg/kg) alone was compared with plasmin plus supplemental AP (5 mg/kg), injected either prior to or after plasmin infusion. We dosed plasmin 8-fold higher than that required for thrombolytic efficacy, based upon our previous observations of its capacity to prolong primary bleeding in greater than 80% of treated animals. Parameters for comparison between treatment groups were: primary bleeding time at timepoints prior to, during, and after experimental infusion, incidence of rebleeding at previously stable hemostatic sites, and quantification of AP, fibrinogen, and factor VIII. Pre-treatment AP doubled the plasma concentration, preserved circulating fibrinogen and factor VIII, and prevented bleeding. Post-plasmin AP corrected the plasmin-associated depletion of AP, but fibrinogen and factor VIII concentrations remained very low, and prolonged bleeding was uncorrected. To test whether AP could be supplemented with coagulation factors in order to arrest bleeding following high dose plasmin, human cryoprecipitate was co-administered. While the fibrinogen and factor VIII levels increased to 200% and 50% of baseline values, respectively, prolonged bleeding persisted. Unexpectedly, administration of human cryoprecipitate led to excess bleeding in association with acute thrombocytopenia and gross hemolysis, presumably mediated by xenographic antibody present in the product. Thus, we find that supplemental AP administered prior to a toxic dose of plasmin prevents coagulation factor consumption and preserves normal hemostasis. Once coagulation factor depletion occurs, AP alone is insufficient to correct the hemorrhagic state that ensues. Clinically, plasmin may be useful as a regional thombolytic agent, and AP supplementation may permit higher dosing as well as protect against accidental overdose. Further studies are needed to determine whether combined AP and coagulation factor repletion may correct the prolonged bleeding induced by massive plasmin infusion.

Distinct dose-dependent effects of plasmin and TPA on coagulation and hemorrhage

AbstractAll thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a direct-acting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages.