Haemostatic safety of a unique recombinant plasmin molecule lacking kringles 2–5

SummaryWe previously demonstrated a significant margin of haemostatic safety for full-length plasmin in comparison with tissue plasminogen activator (t-PA). We now report studies that compare haemostatic safety of full-length plasmin with a novel recombinant plasmin derivative, (Δ K2–5) plasmin, consisting of kringle 1 linked to the serine protease domain of plasmin. Agent was administered intravenously in a randomised, blinded manner in a rabbit model of fibrinolytic haemorrhage. A dose-related decrease in α2-antiplasmin, factor VIII, and fibrinogen followed administration of 1.8, 2.7, 3.7 and 4.6 mg/kg of (Δ K2–5) plasmin, with nadir fibrinogen concentrations of 65%, 40%, 30%, and 0% of initial levels, respectively. Mean primary bleeding time was undisturbed at 1.8 mg/kg (2.2 ± 0.7 minutes), minimally prolonged at 2.7 or 3.7 mg/kg (5 ± 2.9 and 4.4 ± 2.2 minutes), and prolonged at the purposefully toxic 4.6 mg/kg dose (12.8 ± 18.8 minutes). Equimolar amounts of (Δ K2–5) plasmin and full-length plasmin had equal in vitro clot lysis efficacy, but in the bleeding model, (Δ K2–5) plasmin showed better haemostatic competency than full-length plasmin. This safety advantage may be explained by higher residual amounts of plasma fibrinogen in animals given (Δ K2–5) plasmin rather than full-length plasmin. We demonstrate that a unique recombinant plasmin mutant, (Δ K2–5) plasmin, possesses an advantage in hemostatic safety over an equimolar amount of full-length plasmin.

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A Unique Recombinant Plasmin Molecule Lacking Kringles 2-5 Possesses Equivalent Hemostatic Safety as Full-Length Human Plasmin.

Blood ◽

10.1182/blood.v112.11.2028.2028 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2028-2028

Author(s):

Victor J. Marder ◽

Valery Novokhatny ◽

Steve Manyak ◽

Theresa Gruber ◽

Jennifer Hunt ◽

...

Keyword(s):

Animal Model ◽

Factor Viii ◽

Blood Clot ◽

Plasma Concentrations ◽

Full Length ◽

Bleeding Complications ◽

Clot Lysis ◽

Serine Protease Domain ◽

Pre Treatment

Abstract Background: All thrombolytic agents in current clinical use are plasminogen activators (PA). While effective, PA have a downside because of unavoidable and potentially lifethreatening bleeding complications in vulnerable patients. We previously demonstrated a striking margin of hemostatic safety for the “direct-acting” thrombolytic enzyme, plasmin. Now, a novel recombinant derivative of plasmin has been synthesized which lacks kringles 2-5, consisting only of kringle 1 linked to the serine protease domain. Methods: We evaluated escalating dosages of recombinant D (K2-K5) plasmin in a randomized and blinded manner in comparison with molar equivalent dosages of full-length plasmin in an animal model of fibrinolytic hemorrhage. Hemostatic safety was measured by primary bleeding time (PBT) and plasma concentrations of a-2 antiplasmin, factor VIII and fibrinogen. Results: A dose-related nadir in a-2 antiplasmin, factor VIII and fibrinogen followed administration of 4, 6, 8 and 10 mg/kg of D (K2-K5) plasmin, with residual fibrinogen of 65%, 40%, 30% and 0% of initial pre-treatment concentrations. Hemostasis was undisturbed with 4 mg/kg of D (K2-K5) plasmin (mean PBT 2.2 +/- 0.7 min), minimally so at the 6 or 8 mg/kg dosages (5 +/- 2.9 and 4.4 +/- 2.2 min). Hemostasis was markedly abnormal at the purposefully toxic 10 mg/kg dose (12.8 +/- 18.8 min), in association with complete depletion of plasma fibrinogen. Comparison of agents at 10 mg/kg showed no difference, but at 4 mg/kg (2-4-fold more than needed for efficacy), D (K2-K5) plasmin showed slightly less prolongation of PBT (2.2 +/- 0.7 vs 3.3 +/- 2.1 min). Both agents were equally potent for in vitro whole blood clot lysis, indicating that the safety advantage of D (K2-K5) plasmin was not due to decreased fibrinolytic activity. Conclusions: Recombinant D (K2-K5) plasmin has equivalent hemostatic safety as full-length plasmin in an animal model of fibrinolytic hemorrhage and warrants evaluation in clinical thrombotic disease.

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Nanodroplet-mediated catheter-directed sonothrombolysis of retracted blood clots

Microsystems & Nanoengineering ◽

10.1038/s41378-020-00228-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Leela Goel ◽

Huaiyu Wu ◽

Bohua Zhang ◽

Jinwook Kim ◽

Paul A. Dayton ◽

...

Keyword(s):

Drug Delivery ◽

Negative Pressure ◽

Boiling Point ◽

Clot Lysis ◽

Control Group ◽

Peak Negative Pressure ◽

Blood Clots ◽

Tissue Plasminogen ◽

First Time

AbstractOne major challenge in current microbubble (MB) and tissue plasminogen activator (tPA)-mediated sonothrombolysis techniques is effectively treating retracted blood clots, owing to the high density and low porosity of retracted clots. Nanodroplets (NDs) have the potential to enhance retracted clot lysis owing to their small size and ability to penetrate into retracted clots to enhance drug delivery. For the first time, we demonstrate that a sub-megahertz, forward-viewing intravascular (FVI) transducer can be used for ND-mediated sonothrombolysis, in vitro. In this study, we determined the minimum peak negative pressure to induce cavitation with low-boiling point phase change nanodroplets and clot lysis. We then compared nanodroplet mediated sonothrombolysis to MB and tPA mediate techniques. The clot lysis as a percent mass decrease in retracted clots was 9 ± 8%, 9 ± 5%, 16 ± 5%, 14 ± 9%, 17 ± 9%, 30 ± 8%, and 40 ± 9% for the control group, tPA alone, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US groups, respectively. In retracted blood clots, combined ND- and tPA-mediated sonothrombolysis was able to significantly enhance retracted clot lysis compared with traditional MB and tPA-mediated sonothrombolysis techniques. Combined nanodroplet with tPA-mediated sonothrombolysis may provide a feasible strategy for safely treating retracted clots.

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The mechanism of in vitro clot lysis induced by vascular plasminogen activator

Blood ◽

10.1182/blood.v63.6.1331.1331 ◽

1984 ◽

Vol 63 (6) ◽

pp. 1331-1337 ◽

Cited By ~ 16

Author(s):

C Tran-Thang ◽

EK Kruithof ◽

F Bachmann

Keyword(s):

Plasminogen Activator ◽

Whole Blood ◽

Apparent Molecular Weight ◽

Clot Lysis ◽

Initial Concentration ◽

Low Concentrations ◽

Sds Page ◽

Tissue Plasminogen ◽

Low Rate

Abstract The contribution of vascular plasminogen activator (v-PA) to the lysis of whole blood and plasma clots was investigated. v-PA released into the circulation after infusion of deamino-D-arginine vasopressin (DDAVP) was shown to bind quantitatively to plasma clots. Its apparent molecular weight, determined by the SDS-PAGE fibrin-agarose underlay method, was approximately 68,000 daltons, and its activity was quenched by antibodies against human tissue plasminogen activator (t-PA). Clots prepared from post-DDAVP plasma or post-DDAVP whole blood, rich in v- PA, did not lyse when incubated in imidazole buffer or normal plasma, as determined by the release of 125I from radiolabeled clots. However, clots made of v-PA-poor plasma or whole blood, incubated in v-PA-rich plasma, underwent substantial lysis. The concentration of PA in clots incubated in v-PA-rich plasma progressively increased in relation to the initial concentration of v-PA in the surrounding plasma. The results suggest that, at low concentrations of circulating v-PA, a hemostatic plug will lyse at a very low rate. However, when the v-PA concentration in the clot environment is increased, v-PA will accumulate progressively onto fibrin and induce thrombolysis.

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Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid) (PLGA) Magnetic Nanoparticles for Dual Targeted Thrombolytic Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms21082690 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2690 ◽

Cited By ~ 4

Author(s):

Huai-An Chen ◽

Yunn-Hwa Ma ◽

Tzu-Yuan Hsu ◽

Jyh-Ping Chen

Keyword(s):

Magnetic Nanoparticles ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Glycolic Acid ◽

Blood Clot ◽

Recombinant Tissue Plasminogen Activator ◽

Clot Lysis ◽

High Dose ◽

Tissue Plasminogen

Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single –SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area.

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Proteolytic activation of both components of the cation stress–responsive Slt pathway in Aspergillus nidulans

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0049 ◽

2016 ◽

Vol 27 (16) ◽

pp. 2598-2612 ◽

Cited By ~ 9

Author(s):

Laura Mellado ◽

Herbert N. Arst ◽

Eduardo A. Espeso

Keyword(s):

Transcription Factor ◽

Aspergillus Nidulans ◽

Molecular Mechanisms ◽

Mutational Analysis ◽

Full Length ◽

Regulatory Pathway ◽

Protease Domain ◽

Proteolytic Activation ◽

Serine Protease Domain

Tolerance of Aspergillus nidulans to alkalinity and elevated cation concentrations requires both SltA and SltB. Transcription factor SltA and the putative pseudokinase/protease signaling protein SltB comprise a regulatory pathway specific to filamentous fungi. In vivo, SltB is proteolytically cleaved into its two principal domains. Mutational analysis defines a chymotrypsin-like serine protease domain that mediates SltB autoproteolysis and proteolytic cleavage of SltA. The pseudokinase domain might modulate the protease activity of SltB. Three forms of the SltA transcription factor coexist in cells: a full-length, 78-kDa version and a processed, 32-kDa form, which is found in phosphorylated and unphosphorylated states. The SltA32kDa version mediates transcriptional regulation of sltB and, putatively, genes required for tolerance to cation stress and alkalinity. The full-length form, SltA78kDa, apparently has no transcriptional function. In the absence of SltB, only the primary product of SltA is detectable, and its level equals that of SltA78kDa. Mutations in sltB selected as suppressors of null vps alleles and resulting in cation/alkalinity sensitivity either reduced or eliminated SltA proteolysis. There is no evidence for cation or alkalinity regulation of SltB cleavage, but activation of sltB expression requires SltA. This work identifies the molecular mechanisms governing the Slt pathway.

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The effect of 40 kHz ultrasound on tissue plasminogen activator-induced clot lysis in three in vitro models

Ultrasound in Medicine & Biology ◽

10.1016/j.ultrasmedbio.2004.08.028 ◽

2004 ◽

Vol 30 (11) ◽

pp. 1545-1552 ◽

Cited By ~ 18

Author(s):

Marlien Pieters ◽

Rob T. Hekkenberg ◽

Marrie Barrett-Bergshoeff ◽

Dingeman C. Rijken

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

In Vitro Models ◽

Clot Lysis ◽

Tissue Plasminogen

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Clot lysis induced by a monoclonal antibody against alpha 2-plasmin inhibitor

Blood ◽

10.1182/blood.v74.8.2692.bloodjournal7482692 ◽

1989 ◽

Vol 74 (8) ◽

pp. 2692-2697

Author(s):

Y Sakata ◽

Y Eguchi ◽

J Mimuro ◽

M Matsuda ◽

Y Sumi

Keyword(s):

Monoclonal Antibody ◽

Therapeutic Agent ◽

Clot Lysis ◽

Dependent Manner ◽

Plasma Clot ◽

Partial Digestion ◽

Tissue Plasminogen ◽

Dose Dependent ◽

Plasmin Inhibitor

A monoclonal antibody (MoAb) to alpha 2-plasmin inhibitor designated JTPI-1 inhibited antiplasmin activity by interfering with formation of alpha 2-plasmin inhibitor (alpha 2-PI)-plasmin complex. With this MoAb, we observed plasma clot lysis in vitro and evaluated the potential of JTPI-1 to serve as a new therapeutic agent for thrombolysis. After adding 125I-labeled fibrinogen to plasma, clots were made by adding thrombin and calcium and were then resuspended in normal plasma containing various concentrations of JTPI-1. The presence of JTPI-1 enhanced release of the soluble 125I-labeled fibrin degradation fragment from the clots in a dose-dependent manner. With tissue plasminogen activator (t-PA)-depleted plasma, we showed that induction of clot lysis by JTPI-1 was dependent on fibrin-bound endogenous t-PA. Regulation of fibrinolysis initiated on the fibrin surface by fibrin- bound t-PA and plasminogen is mediated by alpha 2-PI cross-linked to fibrin by activated factor XIII. JTPI-1 bound to this cross-linked alpha 2-PI neutralized its activity and induced partial digestion of fibrin by plasmin. This resulted in additional binding of Glu- plasminogen to fibrin during the incubation. When 1.2 mumol/L JTPI-1 and 5 U/mL exogenous t-PA were present in the suspending plasma, the rate of clot lysis was essentially the same as that induced by 60 U/mL exogenous t-PA alone. These results suggest that JTPI-1 may be useful in reducing the amount of t-PA administered for thrombolytic therapy.

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Lysis of intracerebral hematoma with stereotactically implanted tissue plasminogen activator polymers in a rabbit model

Journal of Neurosurgery ◽

10.3171/jns.2006.105.3.424 ◽

2006 ◽

Vol 105 (3) ◽

pp. 424-429 ◽

Cited By ~ 11

Author(s):

Quoc-Anh Thai ◽

Gustavo Pradilla ◽

Federico G. Legnani ◽

Ryan M. Kretzer ◽

Wesley Hsu ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Vinyl Acetate ◽

Rabbit Model ◽

Ethylene Vinyl Acetate ◽

In Vivo Study ◽

Stereotactic Frame ◽

Tissue Plasminogen

Object Currently no adequate surgical treatment exists for spontaneous intracerebral hemorrhage (ICH). Implantable polymers can be used effectively to deliver therapeutic agents to the local site of the pathological process, thus reducing adverse systemic effects. The authors report the use of stereotactically implanted polymers loaded with tissue plasminogen activator (tPA) to induce lysis of ICH in a rabbit model. Methods Ethylene vinyl acetate (EVAc) polymers were loaded with bovine serum albumin (BSA) only or with BSA plus tPA. In vitro pharmacokinetic (three polymers) and thrombolysis (12 polymers) studies were performed. For the in vivo study, 12 rabbits were fixed in a stereotactic frame, and 0.2 ml of clotted autologous blood was injected into the right frontal lobe parenchyma. After 20 minutes, control BSA polymers were stereotactically implanted at the hemorrhage site in six rabbits, and experimental BSA plus tPA polymers were implanted in six rabbits. Animals were killed at 3 days, and blood clot volume was assessed. The pharmacokinetic study showed release of 146 ng of tPA over 3 days. The tPA activity correlated with in vitro thrombolysis. In the in vivo study, the six animals treated with tPA polymers had a mean (± standard error of the mean [SEM]) thrombus volume of 1.43 ± 0.29 mm3 at 3 days, whereas the six animals treated with blank (BSA-only) polymers had a mean (± SEM) thrombus volume of 19.99 ± 3.74 mm3 (p < 0.001). Conclusions Ethylene vinyl acetate polymers release tPA over the course of 3 days. Stereotactic implantation of tPA-loaded EVAc polymers significantly reduced ICH volume. Polymers loaded with tPA may be useful clinically for lysis of ICH without the side effects of systemic administration of tPA.

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Canine Neutrophil Extracellular Traps Enhance Clot Formation and Delay Lysis

Veterinary Pathology ◽

10.1177/0300985817699860 ◽

2017 ◽

Vol 55 (1) ◽

pp. 116-123 ◽

Cited By ~ 10

Author(s):

Unity Jeffery ◽

Dana N. LeVine

Keyword(s):

Autoimmune Diseases ◽

Neutrophil Extracellular Traps ◽

Fibrin Clot ◽

Clot Formation ◽

Clot Lysis ◽

Elastase Activity ◽

Platelet Poor Plasma ◽

Extracellular Traps ◽

Tissue Plasminogen

Autoimmune diseases increase the risk of thrombosis. Neutrophil extracellular traps (NETs) are webs of DNA and protein that may mediate thrombosis in autoimmune diseases. Human and murine studies show NET-releasing neutrophils within a thrombus promote its growth, but it is unclear to what extent NET fragments released into circulation during inflammation are prothrombotic. This study hypothesized that canine NETs promote clot formation and impair lysis even in the absence of neutrophils. NETs were prepared from PMA-stimulated neutrophils and added to fibrinogen and thrombin or to recalcified pooled canine platelet-poor plasma, tissue factor, and tissue plasminogen activator. Clot formation and lysis were measured spectrophotometrically. NETs did not alter fibrin clot formation, but NETs increased maximum clot formation velocity ( P = .001) and delayed lysis ( P = .009) of plasma clots compared with supernatants from nonstimulated neutrophils. DNase digestion of NETs reduced their effect on clot lysis but not maximum clot formation velocity. This suggested impaired lysis was principally mediated by DNA within NETs but that NET proteins were principally responsible for increased speed of clot formation. Previous reports suggested elastase or histones might be responsible for the effect of NETs on clot formation. Elastase activity was greatly reduced by plasma, and addition of histones to plasma did not increase formation velocity, suggesting these proteins were not responsible for increasing maximum formation velocity. This study showed that NETs enhanced clot formation and impaired clot lysis in canine platelet-poor plasma. These in vitro findings suggest both NET proteins and DNA may contribute to thrombosis in inflammatory disease.

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In Vitro Studies on the Fibrinolytic, Thrombolytic and Fibrinogenolytic Properties of a Tissue Plasminogen Activator from Guinea Pig Keratocytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661273 ◽

1985 ◽

Vol 53 (02) ◽

pp. 200-203 ◽

Cited By ~ 4

Author(s):

A Electricwala ◽

R J Ling ◽

P M Sutton ◽

B Griffiths ◽

P A Riley ◽

...

Keyword(s):

Guinea Pig ◽

Human Plasma ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Fibrin Clot ◽

Clot Lysis ◽

Thrombolytic Activity ◽

Tissue Plasminogen ◽

In Vitro Systems

SummaryThe fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37° C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (<50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3–6 hr with the concentration of GPK activator in the range of 1–5 μg/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.

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