Synergistic Effects of Bifunctional Antibodies Against beta3 Integrin on Dissolution of Platelet Thrombus,

Abstract Abstract 3350 HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo. Disclosures: No relevant conflicts of interest to declare.

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Dissolution of arterial platelet thrombi in vivo with a bifunctional platelet GPIIIa49-66 ligand which specifically targets the platelet thrombus

Blood ◽

10.1182/blood-2010-01-264358 ◽

2010 ◽

Vol 116 (13) ◽

pp. 2336-2344 ◽

Cited By ~ 11

Author(s):

Wei Zhang ◽

Yong-Sheng Li ◽

Michael A. Nardi ◽

Suying Dang ◽

Jing Yang ◽

...

Keyword(s):

Blood Flow ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Single Chain ◽

Single Chain Antibody ◽

Antithrombotic Agent ◽

Scfv Library ◽

Platelet Thrombus ◽

12 Lipoxygenase ◽

Carotid Vessels

Abstract Patients with HIV-1 immune-related thrombocytopenia have a unique antibody (Ab) against integrin GPIIIa49-66 capable of inducing oxidative platelet fragmentation via Ab activation of platelet nicotinamide adenine dinucleotide phosphate oxidase and 12-lipoxygenase releasing reactive oxygen species. Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11) capable of inducing fragmentation of activated platelets. In this study, we investigated the in vivo use of A11. We show that A11 does not induce significant thrombocytopenia or inhibit platelet function. A11 can prevent the cessation of carotid artery flow produced by induced artery injury and dissolve the induced thrombus 2 hours after cessation of blood flow. In addition, A11 can prevent, as well as ameliorate, murine middle cerebral artery stroke, without thrombocytopenia or brain hemorrhage. To further optimize the antithrombotic activity of A11, we produced a bifunctional A11-plasminogen first kringle agent (SLK), which homes to newly deposited fibrin strands within and surrounding the platelet thrombus, reducing effects on nonactivated circulating platelets. Indeed, SLK is able to completely reopen occluded carotid vessels 4 hours after cessation of blood flow, whereas A11 had no effect at 4 hours. Thus, a new antithrombotic agent was developed for platelet thrombus clearance.

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A Novel Platelet-Targeting Fibrinolytic Strategy: Endocytosed Urokinase By Megakaryocytes Is Stored in α-Granules and Is Functional In Vivo in a Murine Model of Thrombosis

Blood ◽

10.1182/blood-2019-124501 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3627-3627

Author(s):

Hyunsook Ahn ◽

John Tkaczynski ◽

Khalil Bdeir ◽

Victoria Stepanova ◽

Douglas B. Cines ◽

...

Keyword(s):

Conflicts Of Interest ◽

Ex Vivo ◽

Point Of Care ◽

Ldl Receptor ◽

Single Amino Acid ◽

Platelet Glycoprotein ◽

Single Chain ◽

Injury Model

We have been interested in developing fibrinolytic agents that have a prolonged half-life and can selectively prevent new thrombi from forming without destabilizing established hemostatic clots. We believe that platelet-targeting of urokinase plasminogen activator (uPA) might be able to achieve this goal. Two approaches had been tried in the past: In the first, we had generated chimeric drugs consisting of a platelet-targeting scFvs fused to a thrombin activatable low molecular weight (LMW) uPA (uPA-T). Infused scFv/uPA-T bound to circulating platelets that targeted the fusion within nascent thrombi where significant amounts of thrombin are generated to activate surface-bound scFv/uPA-T. In contrast, mature thrombi are spared because scFv/uPA-T platelets are not activated on the "shell", nor do they penetrate the "core" where thrombin might be present. Murine studies affirmed αIIbβ3-directed scFv/uPA-T provided thromboprophylaxis, but therapeutic doses caused significant thrombocytopenia in murine and baboon models. We therefore considered a second approach: ectopic storage of uPA during megakaryopoiesis. We found that scuPA was stored in platelet α-granules of transgenic mice that ectopically express single-chain uPA (scuPA) and did not cause systemic fibrinolysis. Infusion of such "scuPA platelets" into wildtype mice was highly effective at preventing new thrombi from developing. We now wish to develop this strategy further by taking advantage of ongoing efforts by others to generate in vitro-grown megakaryocytes (Mks) and platelets that can be modified to express uPA near the point-of-care and then infused into patients, bypassing the need to establish uPA-expressing hematopoietic cell lines. We have previously shown that Mks express low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) during their maturation, whereas the platelets that are released do not. We now asked whether in vitro-grown Mks beginning with CD34+ hematopoietic cells would endocytose uPA as seen in other cell types. We show that Mks internalize and store scuPA (scu-Mks, Fig. 1A), as well as LMW uPA and uPA-T (not shown) after overnight incubation. Endocytosed uPA is found within membrane bound structures that partly colocalize with von Willebrand factor (VWF)-positive granules, suggesting uPA is sorted to α-granules (Fig. 1A). Uptake is blocked by receptor-associated protein (RAP), which inhibits endocytosis by LDL receptor family members, including LRP1. We then studied whether platelets with endocytosed scuPA (scuPA-Plts) prevent nascent thrombus development in immunodeficient NOD-scid IL2rγnull (NSG) mice that are also homozygous for VWFR1326H (a single amino acid substitution that switches species selectivity of VWF so that it binds human platelet glycoprotein (GP) Ib/IX receptor rather than mouse GPIb/IX). These mice show a mild bleeding diathesis in a Rose Bengal photochemical carotid artery injury model unless they are infused with human Mks, which we have shown go on to release functional platelets in the recipient animal in its pulmonary capillary bed over the ensuing several hours (Fig. 1B). Thus, when 106 Mks not exposed to uPA were infused so that ~1-10% of circulating platelets were human, thrombi developed in this model; however similar infusion of scuPA-Mks did not occlude (Fig. 1B). In tail clip studies in the same genotypic mice, where the mice were first corrected with human platelets (Fig. 1C) followed 10 min later by scuPA-Mks, rebleeding did not develop when given a similar dose of scuPA-Mks that prevented thrombosis in the photochemical injury model. These studies suggest that Mks internalize biologically relevant concentrations of uPA through a process likely to involve LRP1. Whether ex vivo loading of Mks with uPA can serve as model for point-of-care therapeutics for thromboprophylaxis and diverse other hematologic and non-hematologic indications should be explored. Disclosures No relevant conflicts of interest to declare.

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In Vivo Effect of Suloctidil as an Antiplatelet Agent

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646799 ◽

1979 ◽

Vol 41 (03) ◽

pp. 465-474 ◽

Cited By ~ 7

Author(s):

Marcia R Stelzer ◽

Thomas S Burns ◽

Robert N Saunders

Keyword(s):

Ex Vivo ◽

Antiplatelet Agent ◽

Ratio Method ◽

Platelet Aggregate ◽

Permanent Effect ◽

Platelet Aggregates ◽

5 Ht Uptake ◽

High Doses

SummaryThe relationship between the effects of suloctidil in vivo as an antiplatelet agent and in vitro as a modifier of platelet serotonin (5-HT) parameters was investigated. Suloctidil was found to be effective in reducing platelet aggregates formation in the retired breeder rat as determined using the platelet aggregate ratio method (PAR) with an ED50 of 16.1 mg/kg 24 hours post administration. In contrast to the hypothesis that 5-HT depletion is involved in the anti-aggregatory mechanism of suloctidil, no correlation was found between platelet 5- HT content and this antiplatelet activity. Reduction of platelet 5-HT content required multiple injections of high doses (100 mg/kg/day) of suloctidil. Suloctidil administration for 8 days at 100 mg/kg/day, which lowered platelet 5-HT content by 50%, resulted in no permanent effect on ex vivo platelet 5-HT uptake or thrombin-induced release, nor alteration in the plasma 5-HT level. However, these platelets exhibited a short-lived, significant increase in percent leakage of 5-HT after 30 minutes of incubation. Therefore, suloctidil treatment at high doses may with time result in platelet 5-HT depletion, however this effect is probably not related to the primary anti-aggregatory activity of the drug.

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A 3.5-Second Phenomenon in Haemostasis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649086 ◽

1973 ◽

Vol 30 (02) ◽

pp. 363-370

Author(s):

D Thilo ◽

E Böhm

Keyword(s):

Bleeding Time ◽

Rat Skin ◽

Fibrin Formation ◽

Bleeding Area ◽

Platelet Thrombus ◽

Platelet Aggregates ◽

Primary Platelet ◽

System Mechanism

SummaryExperiments with injury of the abdominal rat skin were carried out to examine the haemostatic system mechanism in vivo after zero to 30 seconds bleeding time. In the bleeding area only a few platelet aggregates could be found with no primary platelet thrombus. After 3.5 second bleeding time the first fibrin strands have been observed at the site of injury. The hypothesis is put forward that there is a very fast reacting haemostatic mechanism which results in the fibrin formation already at 3.5 seconds.

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The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649570 ◽

1993 ◽

Vol 70 (02) ◽

pp. 301-306 ◽

Cited By ~ 51

Author(s):

Linda A Robbie ◽

Nuala A Booth ◽

Alison M Croll ◽

Bruce Bennett

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Fibrin Clot ◽

Clot Lysis ◽

Plasminogen Activator Inhibitor 1 ◽

Dependent Inhibition ◽

Activator Inhibitor ◽

Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

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A fusion protein with improved thrombolytic effect and low bleeding risk

Thrombosis and Haemostasis ◽

10.1160/th09-04-0235 ◽

2009 ◽

Vol 102 (12) ◽

pp. 1194-1203 ◽

Cited By ~ 3

Author(s):

Lisheng Wang ◽

Qinglin Zhang ◽

Yide Qin ◽

Chutse Wu ◽

Xiudong Wang ◽

...

Keyword(s):

Fusion Protein ◽

Anticoagulant Activity ◽

Bleeding Risk ◽

Fibrin Clot ◽

Clot Lysis ◽

Recognition Sequence ◽

Linker Peptide ◽

C Terminus ◽

N Terminus

SummaryTo resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH.We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE andWestern blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin’s anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay.Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.

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My D88-Dependent Interplay Between Myeloid and Endothelial Cells in the Initiation and Progression of Obesity-Associated Inflammatory Diseases

Blood ◽

10.1182/blood.v128.22.sci-44.sci-44 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-44-SCI-44

Author(s):

Xiaoxia Li

Keyword(s):

Insulin Resistance ◽

Endothelial Cells ◽

Adipose Tissue ◽

Systemic Inflammation ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Inflammatory Diseases ◽

Critical Role ◽

Low Grade

Abstract Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

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Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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Effects Of Pentoxifylline, Penbutolol, Prenylamine, Clofibric Acid And Nicotinic Acid On The Release Of Prostacyclin-(PGI2-) Like Activity From Rat Aorta In Vivo And In Vitro

10.1055/s-0038-1652343 ◽

1981 ◽

Author(s):

K U Weithmann ◽

A G Hoechst

Keyword(s):

Cyclic Amp ◽

Nicotinic Acid ◽

Ex Vivo ◽

Synergistic Effects ◽

Rat Aorta ◽

Clofibric Acid ◽

Vessel Walls ◽

Induced Aggregation

Aortas from rats, treated with 5-20 mg/kg of pentoxifylline (pof), penbutolol, prenylamine, clofibric acid or nicotinic acid showed, ex vivo, a significantly higher release of acid labile PGI2-like anti-aggregatory activity compared to controls. This activity could be suppressed by pre-treatment with 2 mg/kg Indomethacin. When incubated with rat aortas in vitro, pof showed a similar stimulatory effect on PGI2-like release, whereas clofibric-and nicotinic acid had no significant effect in this system. Pof and all other drugs mentioned above in therapeutical concentrations had virtually no effect on induced aggregation of human platelets in vitro. However, in the presence of small amounts PGI2 in vitro, inhibition of aggregation and platelet cyclic AMP are enhanced synergistically above the effects of PGI2 and pof individually.We conclude from these experiments that therapeutic doses of all drugs in our study stimulate in vivo the release of PGI2-like activity from vessel walls, thus inhibiting platelet aggregation in vivo. The primary site of action of pof seems to be the vessel wall, whereas the effect of clofibric acid and nicotinic acid on the vessel walls seem to be secondary. The elevation of platelet cyclic AMP levels which generally parallels PGI2-induced inhibition of aggregation might be further enhanced by pof known as an inhibitor of platelet cyclic AMP phosphodiesterase, thus explaining the observed synergistic effects between PGI2 and pof.

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A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma

Blood ◽

10.1182/blood.v75.9.1794.1794 ◽

1990 ◽

Vol 75 (9) ◽

pp. 1794-1800 ◽

Cited By ~ 27

Author(s):

PJ Declerck ◽

HR Lijnen ◽

M Verstreken ◽

H Moreau ◽

D Collen

Keyword(s):

Monoclonal Antibody ◽

Human Plasma ◽

Plasminogen Activator ◽

Fibrin Clot ◽

Clot Lysis ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Plasminogen Activator Inhibitor 1 ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator

Abstract A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

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