Biomedical subjects

J M Stassen

Publications and source records attributed to J M Stassen.

At least 37 records · Page 2Linked to original sources

PubMed1996

Neointima formation in injured hamster carotid artery is effectively prevented by the combination G4120 and quinapril.

The prevention of neointima formation by the tissue selective angiotensin converting enzyme (ACE) inhibitor quinapril and by the combination quinapril/G4120 (a platelet alpha Ub beta 3 and smooth muscle cell alpha v beta 3 antagonist) was investigated in a hamster carotid artery injury model. Quinapril at 10 mg/kg/day reduced neointima formation by about 45%, 1 and 2 weeks after injury to the artery, i.e. significantly better than the non-tissue selective ACE inhibitor captopril at 100 mg/kg/day. Quinapril did not decrease the early smooth muscle cell (SMC) proliferation in the media, but in agreement with its inhibition of the carotid artery ACE activity by 62%, SMC proliferation was reduced by 70% in the newly forming intima. To improve the inhibition of early medial SMC proliferation, quinapril (10 mg/kg/day) was complemented with G4120 (100 micrograms/kg/h). This combined treatment reduced the proliferation of medial SMCs to about 50% throughout the first week following injury, whereas intima SMC proliferation was reduced by 70% throughout treatment. Accordingly, the drug combination reduced neointima formation more potently than each drug separately by 70%. The disruption of medial elastic laminae, observed in the control and G4120 treated group, was consistently reduced when G4120 was complemented with quinapril. Thus, the present study shows in a hamster model of carotid artery injury, that combining drugs that prevent SMC migration and proliferation via different modes of action can lead to the effective prevention of neointima formation.

Analysis of Variance↗

PubMed1995

Antithrombotic effects of thrombolytic agents in a platelet-rich femoral vein thrombosis model in the hamster.

BACKGROUND: The extent and mechanism of the antithrombotic properties of fibrin-selective and non-fibrin-selective thrombolytic agents have not yet been established. METHODS AND RESULTS: The antithrombotic, thrombolytic, fibrinogenolytic, and pharmacokinetic properties of the following substances were determined in hamsters in the absence of conjunctive anticoagulant or antiplatelet therapy: recombinant tissue-type plasminogen activator (rTPA), recombinant single-chain urokinase-type plasminogen activator (rscu-PA), two-chain urokinase-type plasminogen activator (UK), with a rTPA deletion mutant lacking amino acids 6 to 173 and a mutation N184E (K2Pt), a rTPA/rscu-PA chimeric plasminogen activator consisting of amino acids 1 to 3 and 87 to 274 of rTPA and amino acids 138 to 411 of rscu-PA (K1K2Pu), streptokinase (SK), and recombinant staphylokinase (STAR). The anti-thrombotic effect, defined as the intravenous dose required to reduce mural thrombus formation to 50% in a platelet-mediated femoral vein thrombosis model in the hamster, was 6 +/- 1, 5 +/- 2, 1 +/- 0.05, 2.5 +/- 0.2, 0.02 +/- 0.002, 1 +/- 0.09, and 2 +/- 0.3 mg/kg (mean +/- SEM), respectively. The amounts, given as intravenous infusion over 60 minutes that induced 50% clot lysis in a hamster pulmonary embolism model, were 0.18 +/- 0.03, 1.1 +/- 0.05, 0.9 +/- 0.13, 0.34 +/- 0.03, 0.04 +/- 0.003, 0.05 +/- 0.005, and 0.04 +/- 0.001 mg/kg, respectively, indicating that for most thrombolytic agents the antithrombotic dose is much higher than their thrombolytic dose. The fibrinogen levels, measured 40 minutes after bolus injection, were reduced to 50% of baseline with 3.1 +/- 0.2, 2.5 +/- 0.3, 1.2 +/- 0.08, 2.0 +/- 0.14, 1.7 +/- 0.65, 0.54 +/- 0.03, and 1.2 +/- 0.11 mg/kg, respectively. Mean residence times following intravenous bolus injection were: 18 +/- 1, 14 +/- 1, 100 +/- 10, 80 +/- 2, 20 +/- 3, and 34 +/- 5 minutes for rTPA, rscu-PA, K2Pt, K1K2Pu, SK, and STAR, respectively. Regression analysis revealed a significant correlation of the antithrombotic effect with the fibrinogen breakdown (P = .006) but not with the thrombolytic potency or with the mean residence time. CONCLUSIONS: These observations support the hypothesis that thrombolytic therapy with fibrinogen-sparing agents requires the conjunctive use of anticoagulant and/or antiplatelet agents.

Animals↗

PubMed1995

Calin from Hirudo medicinalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions.

Calin from the saliva of the medicinal leech, Hirudo medicinalis, is a potent inhibitor of collagen mediated platelet adhesion and activation. In addition to inhibition of the direct platelet-collagen interaction, we presently demonstrate that binding of von Willebrand to coated collagen can be prevented by Calin, both under static and flow conditions in agreement with the occurrence of binding of Calin to collagen, confirmed by Biospecific Interaction Analysis. To define whether Calin acted by inhibiting the platelet-collagen or the platelet-von Willebrand factor (vWF)-collagen-mediated thrombus formation, platelet adhesion to different types of collagens was studied in a parallel-plate flow chamber perfused with whole blood at different shear rates. Calin dose-dependently prevented platelet adhesion to the different collagens tested both at high- and low-shear stress. The concentration of Calin needed to cause 50% inhibition of platelet adhesion at high-shear stress was some fivefold lower than that needed for inhibition of vWF-binding under similar conditions, implying that at high-shear stress, the effect of Calin on the direct platelet-collagen interactions, suffices to prevent thrombus formation. Platelet adhesion to extracellular matrix (ECM) of cultured human umbilical vein endothelial cells was only partially prevented by Calin, and even less so at a high-shear rather than a low-shear rate, whereas the platelet binding to coated vWF and fibrinogen were minimally affected at both shear rates. Thus, Calin interferes with both the direct platelet-collagen interaction and the vWF-collagen binding. Both effects may contribute to the inhibition of platelet adhesion in flowing conditions, although the former seems to predominate.

Animals↗

PubMed1995

Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters.

Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody-conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose-dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.

Animals↗

PubMed1995

Intraoperative thrombolytic treatment of microarterial occlusion by selective rt-PA infusion.

The development of microvascular thrombosis during replantation surgery or free-flap transfer is generally best treated by identification of the problem, vascular revision, and reanastomosis. It is not unique, however, that surgical measures alone are insufficient or undesirable. Pharmacologic adjuncts are widely used for the prevention and sometimes treatment of microvascular thrombosis in surgical practice, but the benefit of thrombolytic agents, effective in dissolving an already established thrombus, is usually considered counterlevered by the fear of uncontrollable bleeding. However, selective infusion of the drug reduces the risk for systemic complications considerably and may therefore be considered in peripheral microvascular reconstruction. The technique was used successfully in a case of digital revascularization, where an arterial thrombosis was dissolved with the use of recombinant tissue plasminogen activator (rt-PA).

Adult↗

PubMed1995

Differential inhibition with antifibrinolytic agents of staphylokinase and streptokinase induced clot lysis.

The inhibitory effects of antifibrinolytic amino acids on clot lysis induced with recombinant staphylokinase (SakSTAR) or with streptokinase (SK) were evaluated in a human plasma milieu in vitro and in a hamster pulmonary embolism model in vivo. Addition of tranexamic acid to a system composed of 60 microliters 125I-fibrin-labeled plasma clots submerged in 0.5 ml human plasma, caused dose-dependent inhibition of lysis; complete lysis in 120 min required 30 nM SakSTAR or 100 nM SK and was reduced to 50% with 0.015 mM or with 0.07 mM tranexamic acid, respectively. Aprotinin also produced dose-dependent inhibition; lysis with SakSTAR or with SK was reduced to 50% of the control value with 8 KIU/ml or with 10 KIU/ml aprotinin, respectively. Thus, in human plasma in vitro the antifibrinolytic potency of tranexamic acid was 5-fold higher towards SakSTAR than towards SK, whereas that of aprotinin was comparable towards both agents. In hamsters with pulmonary embolism given 0.063 mg/kg SakSTAR or 0.20 mg/kg SK over 30 min, the antifibrinolytic potency of tranexamic acid, administered as a single bolus injection or as a bolus injection followed by continuous infusion, was 8- to 10-fold higher towards SakSTAR than toward SK (50% reduction of clot lysis with SakSTAR at 12.5 mg/kg, as compared to 100-150 mg/kg with SK). In contrast, aprotinin was equipotent towards SakSTAR and SK (50% reduction of clot lysis with 2,000 to 2,700 KIU/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Characterisation of a novel series of aprotinin-derived anticoagulants. I. In vitro and pharmacological properties.

Previous investigations have indicated that interference with the initial level of the blood coagulation may lead to effective antithrombotic therapy. Recently a series of potential coagulation inhibitors derived from bovine pancreatic trypsin inhibitor (BPTI, aprotinin) was described. We have determined their inhibition constants, effects on coagulation assays, effects in an in vitro human thrombosis model and pharmacological profiles in hamsters. The aprotinin-derived analogues (4C2, 7L22, 5L15, 6L15, 5L84) showed significantly increased inhibitory activity towards factor Xa, factor VIIa-tissue factor (TF) complex, factor XIa and plasma kallikrein or a combination of them, and a significantly decreased plasmin inhibition as compared to aprotinin. In the coagulation assays, 4C2 and 7L22 mainly inhibited factor Xa, 5L15 and 6L15 inhibited factor VIIa-TF complex and 5L84 inhibited factor Xa, factor VIIa-TF complex and the contact activation. In flow chamber experiments with human blood 7L22, 5L15, 6L15, 5L84 and rTAP significantly inhibited fibrin formation and platelet deposition on extracellular matrix from phorbol ester stimulated human endothelial cells both under high and low shear stress and in the presence of low molecular weight heparin. The pharmacological profiles of the aprotinin analogues and rTAP with a mean residence time of 64 to 140 min were not significantly different. Modification of an aprotinin analogue with PEG (5L15-PEG) resulted in a 10-fold decrease of the inhibition constant for the factor VIIa-TF complex and in a significant prolongation of the secondary half-life, while the initial half-life was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Characterisation of a novel series of aprotinin-derived anticoagulants. II. Comparative antithrombotic effects on primary thrombus formation in vivo.

Upon vascular damage platelet activation and blood coagulation are initiated. Interference at the initial level of the activation of the coagulation cascade can result in effective inhibition of thrombus formation. The in vivo antithrombotic properties of a series of bovine pancreatic trypsin inhibitor mutants (BPTI, aprotinin) 4C2, 7L22, 5L15, 5L15-PEG, 6L15 and 5L84, as described in the accompanying paper, with a combined inhibitory activity on factor Xa, factor VIIa-tissue factor complex, factor XIa and plasma kallikrein were compared to rTAP, r-hirudin, heparin and enoxaparin in a platelet rich thrombosis model in hamsters. Platelet dependent thrombus deposition was quantified by dedicated image analysis after transillumination of the femoral vein to which a standardised vascular trauma was applied. After increasing intravenous bolus injections all tested agents, except for aprotinin, induced a dose dependent decrease of thrombus formation and a concomitant prolongation of the aPTT. From the linear correlation between these two parameters it was found that 5 out of the 6 tested aprotinin analogues, rTAP and r-hirudin completely inhibited thrombus formation at a therapeutical (2- to 3-fold) aPTT prolongation while 4C2, heparin and enoxaparin only inhibited thrombus formation for 40 to 50 percent at a 2-fold aPTT prolongation. Based on the calculated IC50 values for thrombus formation rTAP was found to be the most active compound in this model. It is concluded that acceptable interference at the initial level of the blood coagulation, e.g. within a therapeutical aPTT prolongation, can significantly inhibit platelet deposition at a site of vascular injury.

Animals↗

PubMed1995

Prolonged inhibition of acute arterial thrombosis by high dosing of a monoclonal anti-platelet glycoprotein IIb/IIIa antibody in a baboon model.

The in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dose-finding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrombotic effect was stronger 48 h after a dose of 0.3 mg/kg administration than on the day of treatment with 0.1 mg/kg, despite the fact that comparable numbers of GPIIb/IIIa receptors were occupied on resting platelets. We postulate that with the high dose of MA-16N7C2 and after an extended period, occupied GPIIb/IIIa may be internalised by the platelets. Upon platelet activation, these receptors become reexposed but are unable to participate in thrombus formation. This is in contrast to unoccupied internal GPIIb/IIIa receptors early after a low dose of MA-16N7C2.

Amino Acid Sequence↗

PubMed1995

Fast and reproducible vascular neointima formation in the hamster carotid artery: effects of trapidil and captopril.

Neointima formation was induced in the hamster carotid artery by mechanical intraluminal injury with a catheter covered with roughened dental cement, Neointimal thickening occurred as early as 7 days after denudation and further increased during the next 1 to 2 weeks. Proliferation indices of smooth muscle cells (SMCs) showed the highest proportion of proliferating cells in the media and neointima respectively 1 and 5 days after the vascular injury. Transmission and scanning electron microscopy of damaged carotid artery sections as well as immuno-histochemical stainings of von Willebrand factor (vWF) confirmed that reendothelialization was progressive and already complete on day 14, at which time the neointima formation was almost complete. In order to pharmacologically characterize this model further, the effects on neointima formation of trapidil (triazolopyrimidine), a platelet-derived growth factor (PDGF) antagonist, and captopril, an angiotensin converting enzyme inhibitor, were investigated. Trapidil administered orally twice daily at total doses of 25, 50 and 100 mg/kg/day, started 3 days prior to infliction of injury and up to 7 or 14 days after the catheterization, significantly reduced neointima formation. Captopril administered orally three times daily at a total dose of 100 mg/kg/day, equally reduced neointima formation, with 100 mg/kg/day trapidil being more effective than 100 mg/kg/day captopril 7 days after injury. When the treatment by either one of these drugs was arrested on day 7, neointima formation resumed quickly. The hamster appears to be a small, reproducible and fast model for the study of SMC proliferation, requiring only relatively small amounts of experimental drugs. The model furthermore is sensitive to substances known to reduce neointima formation in other animal models.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1994

Contribution of platelets and the vessel wall to the antithrombotic effects of a single bolus injection of Fab fragments of the antiplatelet GPIIb/IIIa antibody 7E3 in a canine arterial eversion graft preparation.

The contribution of platelets and the vessel wall to the antithrombotic effects of a single intravenous bolus injection of 0.8 mg/kg Fab fragments of the monoclonal antiplatelet glycoprotein IIb/IIIa receptor antibody 7E3 (7E3-Fab), combined with continuous heparin anticoagulation (100 U/kg bolus and 50 U/kg per hour), was studied in a canine preparation consisting of an everted (inside out) carotid arterial segment that had been inserted into a transected femoral artery. In all 6 control dogs without antibody, persistent or transient eversion graft occlusion occurred during an initial 2-hour observation period, and 5 of the 6 grafts were occluded at 24 hours. In 6 dogs given 7E3-Fab 24 hours before receiving an everted carotid artery segment from a donor dog, cyclic occlusion and reflow occurred in all dogs, whereas the grafts were patent at the end of a 2-hour observation period in 5 of the 6 dogs (P = .056 versus control). When transferred back to the donor dogs, the patient eversion segments showed brief periods of cyclic occlusion and reflow within 2 hours in 3 of 5 dogs (P = .034 versus control), whereas all of the 5 eversion segments were patent at 24 hours (P < .005 versus control).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation.

BACKGROUND: RGD-containing peptides are able to prevent binding of ligands to certain integrins such as alpha IIb beta 3 (glycoprotein IIb/IIIa) and alpha v beta 3 and as such are inhibitors for platelet aggregation and smooth muscle cell migration, both of which are involved in neointima formation. METHODS AND RESULTS: Hamster carotid arteries were damaged, and neointima formation was determined at different time points. G4120, a cyclic RGD-containing peptide, was administered continuously intravenously by an implanted osmotic pump. Neointima formation was inhibited dose dependently. The inhibition was strongest when treatment was started before the vascular injury and continued for the full observation period. Treatment started after the damage and maintained until neointima assessment or started before and stopped earlier was less effective. CONCLUSIONS: Inhibition of integrin function by an RGD-containing peptide results in reduction of the development of a neointima. This effect is due both to an early event, which could be due to inhibition of secretion of PDGF by the platelets with blocked alpha IIb beta 3, and to a late event, possibly by interference with smooth muscle cell alpha v beta 3.

Animals↗

PubMed1994

On the immunogenicity of recombinant staphylokinase in patients and in animal models.

Streptokinase and staphylokinase, the presently available thrombolytic agents of bacterial origin, are immunogenic in man; their use may cause allergic reactions and/or refractoriness to renewed administration. Infusion of 2 to 10 mg of recombinant staphylokinase (STAR) in 20 patients with acute myocardial infarction or peripheral arterial occlusion induced IgG-related neutralizing activity in plasma with a lag phase of 10 to 12 days, from a baseline of 0.2 +/- 0.06 microgram STAR neutralized per ml plasma (mean +/- SEM) to a maximum of 30 +/- 6.2 micrograms/ml after 3 to 9 weeks, which persisted at a level of 14 +/- 5.8 micrograms/ml after 18 months (n = 4). In 4 baboons with a 125I-fibrin labeled clot in an extracorporeal arteriovenous loop, i.v. administration of 63 micrograms/kg STAR over 1 h, repeated at weekly intervals, induced a progressive increase of STAR-neutralizing activity (from 0.05 +/- 0.1 microgram/ml at baseline to 4.8 +/- 1.5 micrograms/ml at week 6), which was paralleled by a reduction of in vivo clot lysis (from 60 +/- 7% to 8 +/- 3%). After temporary discontinuation of STAR-administration, neutralizing activity reverted to baseline within 7 weeks, whereafter the sensitivity of in vivo clot lysis to STAR was restored. In rabbits, i.v. administration of 250 micrograms/kg STAR over 1 h, repeated at weekly intervals, also induced a progressive increase of STAR-neutralizing activity (from 0.5 +/- 0.2 microgram/ml at baseline to 6.4 +/- 1.1 micrograms/ml at week 6), which was paralleled by a reduction of in vivo clot lysis (from 68 +/- 3% to 31 +/- 7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Comparative thrombolytic properties of tissue-type plasminogen activator and of a plasminogen activator inhibitor-1-resistant glycosylation variant, in a combined arterial and venous thrombosis model in the dog.

rt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagenized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent. The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 +/- 100% versus 140 +/- 40% within the 2 h observation period per mg of compound administered per kg body weight (mean +/- SEM, p = 0.004) and a significantly lower dose of 0.08 +/- 0.01 versus 0.21 +/- 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Antithrombotic effects and bleeding time prolongation with synthetic platelet GPIIb/IIIa inhibitors in animal models of platelet-mediated thrombosis.

Cyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-alpha-aspartyl- cyclic (1-->5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-propyl-L-arginyl-glycyl-L-alpha- aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1-->9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor. The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 micrograms/kg, ex vivo ADP-induced platelet aggregation with ID50 of 10 micrograms/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 +/- 9 to 1,100 +/- 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 micrograms/kg, ex vivo platelet aggregation with an ID50 of 50 micrograms/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 micrograms/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induced ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 +/- 0.4 to 12 +/- 2 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Comparative effects of enoxaparin and heparin on arterial and venous clot lysis with alteplase in dogs.

The effects of Enoxaparin with a specific anti-thrombin (anti-IIa) activity of 32 U/mg and a specific anti-factor-XA (anti-Xa) activity of 96 U/mg, and of heparin with a specific anti-IIa and anti-Xa activity of 192 U/mg, on thrombolysis with alteplase (Actilyse) were compared in a randomized blinded study using a combined arterial and venous thrombosis model in the dog. All dogs received an intravenous bolus of 5 mg/kg lysine-acetyl salicylate and 0.5 mg/kg alteplase over 60 min. Twenty-eight dogs were randomly assigned to seven treatment groups: placebo, Enoxaparin 1.5, 3 or 6 mg/kg, or heparin 0.5, 1 or 2 mg/kg, given as a 50% intravenous bolus and a 50% infusion over 2 h. Steady-state plasma levels ranged from 0.37 to 1.0 anti-IIa U/ml and 0.9 to 3.1 anti-Xa U/ml for Enoxaparin and from 0.4 to 2.3 anti-IIa U/ml and 0.42 to 3.2 anti-Xa U/ml for heparin. The activated thromboplastin time with 6 mg/kg Enoxaparin prolonged to 94 +/- 19 s and with 2 mg/kg heparin to > 150 s. The time to reflow was 120 +/- 36 min with placebo, 19 +/- 5 min with 6 mg/kg Enoxaparin (p = 0.03 vs control), and 22 +/- 5 min with 2 mg/kg of heparin (p = 0.03 vs control). Arterial patency, expressed in min reflow during the 180 min observation period correlated significantly with the dose of anticoagulant given (r = 0.73, p = 0.003 for Enoxaparin and r = 0.61, p = 0.012 for heparin).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody fused to single-chain urokinase.

The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single-chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady-state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody-targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.

Amino Acid Sequence↗

PubMed1993

Thrombolytic profiles of clot-targeted plasminogen activators. Parameters determining potency and initial and maximal rates.

BACKGROUND: Targeting of plasminogen activators to the thrombus by means of fibrin-specific monoclonal antibodies may enhance their thrombolytic potency. The kinetics of clot binding of two human fibrin-specific monoclonal antibodies (MA-12B3 and MA-15C5) and of clot lysis with their chemical 1:1 stoichiometric complexes with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) (rscu-PA/MA-12B3 and rscu-PA/MA-15C5) were determined in hamsters and rabbits. Thrombolytic potencies, maximal rates of clot lysis, and the duration of the lag phases before clot lysis of the antibody/rscu-PA conjugates were compared with those of rscu-PA and tissue-type plasminogen activator (rt-PA). METHODS AND RESULTS: Bolus injection of 7.5 micrograms of 125I-labeled antibody in rabbits with an extracorporeal arteriovenous loop containing a 0.3-mL human plasma clot produced clot-to-blood ratios of 6.6 +/- 1.0 (mean +/- SEM) for MA-12B3 and 1.1 +/- 0.15 for MA-15C5 (p < 0.001 versus MA-12B3) within 6 hours. Progressive digestion of the clot did not alter the binding of MA-12B3 but resulted in as much as a 10-fold increase of the binding of MA-15C5. The conjugates infused intravenously over 90 minutes in hamsters with a human plasma clot in the pulmonary artery produced dose-related in vivo clot lysis. Thrombolytic potencies (maximal slope of the percent lysis versus dose in milligrams of u-PA equivalent per kilogram body weight) were 2,500 +/- 440 for rscu-PA/MA-12B3, 3,600 +/- 640 for rscu-PA/MA-15C5 (p = NS vs. rscu-PA/MA-12B3), 60 +/- 8 for rscu-PA (p < 0.001 versus both conjugates), and 380 +/- 66 for rt-PA (p < 0.001 versus both conjugates). The plasma clearances of the conjugates were fourfold to sixfold slower than those of rscu-PA and rt-PA. Maximal rates of clot lysis, determined by continuous external radioisotope scanning over the thorax, were 0.90 +/- 0.13%, 0.91 +/- 0.17%, 0.84 +/- 0.12%, and 1.1 +/- 0.16% lysis per minute for rscu-PA/MA-12B3, rscu-PA/MA-15C5, rscu-PA, and rt-PA, respectively; these maximal rates were obtained with 0.016, 0.016, 1.0, and 0.25 mg/kg, respectively, and were associated with minimal lag phases of 18 +/- 3.2, 28 +/- 4.9, 34 +/- 3.7, and 25 +/- 3.9 minutes, respectively. CONCLUSIONS: The thrombolytic potency of the rscu-PA/antifibrin conjugates is determined by their clearance, as well as by rate and extent of initial binding to clots and by changes in binding during clot lysis. Clot targeting of rscu-PA with fibrin-specific antibodies increases its thrombolytic potency but does not alter the maximal rate or the minimal lag phase of clot lysis. These parameters appear to be independent of the nature of the plasminogen activator and of targeting.

Animals↗