(Circulation. 1997;96:941-948.)
© 1997 American Heart Association, Inc.
Articles |
Reduction in Stent and Vascular Graft Thrombosis and Enhancement of Thrombolysis by Recombinant Lys-Plasminogen in Nonhuman Primates
From the Division of Hematology-Oncology, Department of Medicine, Yerkes Regional Primate Research Center, Emory University School of Medicine, Atlanta, Ga, and Vessel Wall Biology, NovoNordisk A/S, Genetofte, Denmark (L.C.P., U.H.).
Correspondence to Laurence A. Harker, MD, Division of Hematology and Oncology, Emory University School of Medicine, 1639 Pierce Dr, Room 1003, Atlanta, GA 30322.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background To enhance thrombolytic responses without increasing hemorrhagic risks, the antithrombotic effects of recombinant Lys-plasminogen (r-LysPgn), a prothrombolytic plasminogen intermediate, were examined in baboon models of thrombus formation and dissolution.
Methods and Results The dose-response effects of r-LysPgn, alone or in combination with subthreshold dosing of tissue plasminogen activator (TPA), were measured with respect to the accumulation of 111In-labeled platelets and 125I-fibrin in thrombus forming on endovascular metallic stents or thrombogenic segments of vascular graft interposed in exteriorized long-term arteriovenous (AV) femoral shunts. Thrombolytic losses have also been determined for preformed, stable, 111In-platelet– and 125I-fibrin–labeled graft thrombus and corresponding propagated thrombotic tails, together with changes in blood tests of thrombosis, thrombolysis, and hemostasis. Bolus intravenous r-LysPgn in escalating doses (2, 4, or 8 mg/kg) increased circulating plasminogen levels in a dose-dependent manner, was removed by log-linear clearance with a T50 of 120 minutes, and reciprocally decreased the accumulating thrombus on metallic stents and segments of vascular graft (P<.001 in all cases for 8-mg/kg doses). r-LysPgn also impaired platelet aggregatory responses to physiological agonists in vitro but not ex vivo. Prethrombosis administration of low-dose r-LysPgn (2 mg/kg) greatly enhanced the lysis of radiolabeled nonoccluding thrombus by a subthreshold dose of TPA (0.1 mg/kg) compared with TPA-only controls (P=.03).
Conclusions Elective bolus injections of r-LysPgn before stent deployment decrease the amount of thrombus formed without compromising hemostasis by facilitating endogenous TPA thrombolysis. r-LysPgn may provide effective and safe antithrombotic therapy for interventional vascular procedures.
Key Words: plasminogen • platelets • thrombolysis • plasminogen activators
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Thrombotic occlusion of coronary and cerebral arteries at sites of stenosing atherosclerotic disease causes heart attacks and strokes.1 2 3 Rapid restoration of arterial patency by early thrombolytic therapy rescues ischemic tissues.4 5 6 7 8 9 10 Unfortunately, such treatment is marred by (1) failure to achieve reperfusion in 20% of patients receiving recommended clinical dosing; (2) abnormal bleeding, in particular devastating hemorrhagic stroke in 1% of patients; and (3) rethrombosis in 5% to 15% of patients who achieve initial reperfusion.4 5 6 7 8 9 10 Although clinical efficacy has not been improved by either increasing thrombolytic dosing or coadministering adjunctive antithrombotic treatment, these therapeutic modifications predictably increase the frequency and severity of hemorrhagic complications.11 12 13 14 15 Thus, strategies are needed for enhancing thrombolytic responses without increasing the risk of hemorrhage.
Physiological fibrinolysis involves the proteolytic conversion of plasminogen to plasmin by TPA, followed by plasmin-mediated catalytic degradation of fibrin.16 The rate at which TPA cleaves plasminogen is accelerated 1000-fold in the presence of fibrin.3 Further fibrinolytic enhancement is achieved when Lys-plasminogen substitutes for Glu-plasminogen as the substrate for TPA. Lys-plasminogen is a truncated intermediate generated by limited plasmin cleavage of native Glu-plasminogen.17 18 Lys-plasminogen binds more avidly to fibrin and is more readily activated by TPA or UPA than full-length Glu-plasminogen.3 16 In experimental animals, injections of r-LysPgn amplify fibrinolysis by TPA or UPA.19 20 21 In the present study, we used quantitative nonhuman primate models of thrombus formation and dissolution to explore the hypothesis that administering r-LysPgn augments thrombolysis and diminishes new thrombus formation without impairing hemostasis. r-LysPgn expressed by mammalian cells was purified by techniques reported previously.22
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Baboon Model of Thrombolysis, Thrombosis, and Hemostasis
In the baboon model of thrombus formation and dissolution used in this study, quantitative, reproducible, nonoccluding, platelet-rich thrombi with fibrin-rich propagated thrombotic tails are formed on thrombogenic segments of vascular graft interposed in long-term AV shunts under physiological flow conditions for 75 minutes without systemic anticoagulation. The forming thrombus incorporates circulating prelabeled autologous 111In-labeled platelets and homologous 125I-fibrin(ogen). The effects of thrombolytic agents are subsequently evaluated on preformed, isotopically labeled, heparin-stabilized thrombus. Changes in relevant blood tests of fibrinolysis, thrombosis, and hemostasis are also measured, including template BT estimation of platelet hemostatic function.23
Eighteen normal juvenile male baboons weighing 9 to 11.5 kg and bearing long-term exteriorized AV femoral shunts were used in these studies. Before experimentation, all animals were observed to be disease free for at least 3 months. Long-term exteriorized AV angioaccess shunts were surgically placed between the femoral artery and vein, with ketamine hydrochloride (20 mg/kg) used for induction, 1% halothane by endotracheal tube for anesthetic maintenance, and buprenorphine (0.01 mg/kg every 8 hours as needed) for postoperative analgesia. Ketamine hydrochloride (5 to 20 mg/kg IM) was used for subsequent short-term immobilization in experimental procedures. All procedures were approved by the Institutional Animal Care and Use Committee (Emory University) in compliance with National Institutes of Health guidelines (Guide for the Care and Use of Laboratory Animals, 1985), Public Health Service policy, the Animal Welfare Act, and related university polices.
The long-term AV shunts were composed of silicone rubber tubing of 3.0-mm ID (Silastic, Dow Corning Corp). The arterial and venous arms of the shunt were connected with a 1-cm length of polytetrafluoroethylene (Teflon) tubing (2.8-mm ID). All materials were sterilized by autoclaving before surgical placement. These long-term AV shunts do not detectably activate platelets or fibrinogen.24 25
Nonocclusive thrombi were formed over a period of 75 minutes in the exteriorized AV shunts of awake animals by placement of 2-cm-long, 4-mm-ID thrombogenic segments of uncrimped Dacron vascular grafts (Bioknit, C.R. Bard, Inc) in the AV shunts and control of the blood flow at 40 mL/min. The segments were rendered impervious to blood leakage by external wrapping in Parafilm (American Can Company) and 5.3-mm-ID "heat-shrink" Teflon tubing. Connections were constructed to ensure that the devices were isodiametric and suitable for incorporation into the AV shunts. Because blood flows of 40 mL/min through the AV shunts produced venous-type shear rates, thrombus forming on segments of vascular graft developed venous-type fibrin-rich propagated thrombotic tails extending downstream from the vascular graft.23 26 27 28
Thrombi formed over a period of 75 minutes were maintained relatively constant without occlusion during the ensuing 60 minutes of study by administration of heparin as a bolus (100 U/kg IV) followed by continuous infusions of additional heparin totaling 100 U/kg administered over 60 minutes, as reported previously.23 When TPA was infused, an initial bolus consisting of 10% of the dose was injected, and the remainder was infused continuously over 60 minutes (concurrently with heparin). Lysis of the isotopically labeled, preformed thrombus was measured by gamma camera imaging throughout the 60 minutes of TPA infusion.23
Stent thrombosis was produced by deploying stainless steel endovascular stents (3.5 mm) in the exteriorized long-term AV shunt flowing at 100 mL/min and measuring 111In-platelet deposition, 125I-fibrin accumulation, and changes in thrombosis blood tests. The stainless steel stents (316SL) were a gift from Johnson & Johnson Interventional Systems. They were mounted on sterile water–filled noncompliant Duralyn coronary angioplasty balloons (Cordis Corp). The stents were manually crimped on the deflated balloon and inserted into a 3.3-mm-ID, 20-cm-long segment of silicone rubber tubing (Technical Products Inc). The balloon was inflated three times to 10 atm pressure to achieve maximal apposition of the stent struts with the tubing wall. The shunt tubing was then filled with sterile saline to remove potentially confounding air bubbles from the surface of the stent and interposed in the exteriorized long-term AV femoral shunt, and blood flow at 100 mL/min was initiated and maintained for 60 minutes.
Blood samples for tests of thrombolysis, thrombosis, and hemostatic function were collected in citrate, EDTA, or anticoagulant/inhibitor mixture (1) before the thrombogenic devices were placed (0 minutes), (2) before the administration of heparin was begun (75 minutes), (3) at the end of the test infusion (135 minutes), and (4) at the conclusion of the study (165 minutes). Samples were coded and tested in a "blinded" manner.27 28 29
Recombinant Human Lys-Plasminogen
r-LysPgn was expressed by transfected baby hamster kidney
cells as described previously.22 r-LysPgn was purified
from the culture medium by means of ion exchange
chromatography and affinity
chromatography on lysine-Sepharose. Medium adjusted to
pH 5.0 was applied to S Sepharose equilibrated with 0.04 mol/L NaCl,
0.02 mol/L NaH2PO4, 0.02% Tween 80, and 3 mg/L
BPTI, pH 5.0. Washing with 0.12 mol/L NaCl, 0.02 mol/L
NaH2PO4, 0.02% Tween 80, and 3 mg/L BPTI, pH
5.5, was followed by elution of r-LysPgn with 0.18 mol/L NaCl, 0.02
mol/L NaH2PO4, 0.02% Tween 80, 3 mg/L BPTI,
and 3 mmol/L 6-amino hexanoic acid, pH 5.5. The eluate was
adjusted to pH 4.5 and applied to an S Sepharose column equilibrated
with 0.18 mol/L NaCl, 0.02 mol/L NaH2PO4, and
0.02% Tween 80, pH 4.5. After a washing with the same buffer, r-LysPgn
was eluted with 0.02 mol/L NaH2PO4 and 0.02%
Tween 80, pH 7.4. Further purification was performed by affinity
chromatography on lysine-Sepharose.30
After elution with 0.1 mol/L NaH2PO4, pH 7.4,
the 10-mmol/L 6-amino hexanoic acid in the eluate was removed by
ultrafiltration against distilled water adjusted to pH 4.0 with HCl.
Finally, r-LysPgn was obtained as lyophilized protein.
r-LysPgn was administered in doses of 2, 4, and 8 mg/kg IV 15 minutes before thrombogenic devices were interposed in AV shunts. In subsequent experiments, 2 mg/kg r-LysPgn was injected at time 0, and TPA (0.1 mg/kg) was begun at 75 minutes and continued for 60 minutes. In follow-up experiments, 2 mg/kg r-LysPgn was injected after, as opposed to before, the formation of vascular graft thrombus at 75 minutes, followed by 60 minutes of 0.1-mg/kg TPA infusion. This dose of TPA was subthreshold, ie, not sufficient to produce detectable fibrinolytic effects in this model.23 The test agents and doses were studied in animals by random sequence.
Measurements of Thrombus Formation and Dissolution
Autologous platelets were labeled with 1 mCi
111In oxine (111In) as previously
described23 26 27 28 and reinjected at least 1 hour before
the thrombogenic segments of vascular graft were interposed. Baboon
fibrinogen was purified by ß-alanine precipitation and labeled with
125I by the ICl method described previously.27
A 5-µCi dose of 125I-fibrinogen was injected
intravenously 10 minutes before the thrombogenic segments
were introduced.
Images of the segment of proximal vascular graft thrombi and distal propagated thrombotic tail and of the stent-containing segments were acquired separately with a General Electric 400T MaxiCamera and stored and analyzed with a Medical Data Systems A3 image processing system (Medtronic) interfaced with the camera. The low-energy peak (172 keV) of 111In was imaged with a 10% window, and dynamic images were acquired at 5-minute intervals. Immediately after each dynamic study, a 5-mL whole-blood standard was imaged in an identical configuration.23 Thrombus radioactivity was counted and analyzed as regions of interest for the stents and for the platelet-rich thrombus attached to the 2-cm segment of vascular graft (8x10 pixels) and separately for the fibrin-rich propagated tail for 20 cm immediately distal to the vascular graft (80x10 pixels). The total number of deposited platelets in each region was calculated by dividing the deposited platelet activity (cpm) by the circulating blood activity (cpm/mL) and multiplying by the circulating platelet count (platelets/mL). Radioactivity referred to platelet activity only, the small amount of nonplatelet radioactivity having been corrected for.
Fibrin in platelet-rich and fibrin-rich component thrombi was determined after the completion of the experiments by removal of the segments containing the stents or vascular graft and corresponding propagated thrombotic tails for counting of 125I-fibrin radioactivity 30 days later when the 111In activity had decayed. Total fibrin accumulated was calculated by dividing the deposited 125I activity (cpm) by the clottable fibrinogen activity (cpm/mL) and multiplying by the plasma fibrinogen level (mg/mL).26 27
Laboratory Studies
Platelet, erythrocyte, and total leukocyte counts were
performed on whole blood collected in 2 mg/mL disodium EDTA with a
Serono Baker model 9000 whole-blood
analyzer.26 27 31 32 33 34 35 36 37
Platelet aggregation was determined within 1 hour of drawing blood with a Chrono-Log aggregometer by recording the increase in light transmission through a stirred suspension of PRP maintained at 37°C. PRP and platelet-poor plasma were prepared by differential centrifugation, as previously described.35 38 The platelet count in the PRP was adjusted to 300x103/µL. Percent aggregation was calculated linearly between the optical densities of platelet-poor plasma and PRP. ADP (Sigma Chemical Co), collagen (Nycomed Arzneimittel), and TRAP1-6 (Peninsula Laboratories) were added at doses spanning the range of responsiveness. The results were plotted and expressed as the agonist concentration that induced half-maximal aggregation (AC50).27 35 38 PRP was incubated for 15 minutes in polypropylene tubes with or without r-LysPgn (125 µg/mL), a plasma concentration similar to that obtained in baboons after injection of 8 mg/kg r-LysPgn.
Template BT measurements were performed at baseline and at 135 minutes when heparin was discontinued. BT testing was carried out on the shaved volar surface of the forearm as previously described in nonhuman primates.26 27 31 32 33 34 35 36 In baboons, the BT reliably and reproducibly measures overall platelet hemostatic plug-forming capability.26 27 31 32 33 34 35 36 37 39 40
Blood for measuring TAT, plasminogen, and D-dimer was collected in 1/10 vol 3.8% trisodium citrate. FPA was measured in blood collected in EDTA, heparin, aprotinin, and D-Phe-Pro-Arg chloromethylketone (PPACK).23 39 40 Platelet-free plasma was prepared by centrifugation at 10 000g for 5 minutes and stored frozen until immunometric assays were performed. We have previously shown that antibodies directed against human plasminogen, FPA, and D-dimer correspondingly cross-react with baboon plasminogen, FPA, and D-dimer.23 We therefore used commercially available ELISA kits for measuring plasminogen (Spectrolyse/Plasminogen, Biopool), FPA (IMCO), and fibrin degradation products containing the D-dimer configuration (Dimertest EIA, American Diagnostica). Plasma fibrinogen concentrations were measured as thrombin-clottable protein by the method described previously.41 42 43
Statistical Analysis
Data are presented as mean±SD. Student's t
test for paired or unpaired data was used where data were normally
distributed. Otherwise, Mann-Whitney nonparametric
analysis was used. ANOVA was used for factorial
analysis of variance and covariance. A value of
P
.05 was considered to be the estimate of statistical
significance.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Clearance of Injected r-LysPgn
Bolus injections of r-LysPgn increased circulating concentrations of plasminogen in a dose-dependent manner (Table 1
). Fifteen minutes after 8 mg/kg r-LysPgn was
injected, the plasminogen concentration was increased to
219±34 µg/mL versus the baseline of 102±27 µg/mL
(P=.001). Plasma plasminogen levels gradually
fell after r-LysPgn was injected. The disappearance pattern was
obtained by plotting the difference between plasma
plasminogen levels from baseline values over time (Fig 1). Based on the regression analysis of the
decremental changes after injection of 8 mg/kg r-LysPgn, the
T50 clearance estimate of injected r-LysPgn in baboons was
120 minutes (Fig 1). During the interval when
thrombolytic effects were being assessed in the
experiments (from 75 to 135 minutes), injections of 8 mg/kg r-LysPgn
maintained the circulating concentrations of plasminogen
approximately twofold over baseline, consisting of approximately
equivalent amounts of r-LysPgn and native Glu-plasminogen
(Table 1).
|
|
) are plotted over time. Disappearance pattern
of injected r-LysPgn is log-linear with a T50 disappearance
of 120 minutes. Error bars represent ±1 SD.
Thrombus Formation on Vascular Grafts in Untreated Control
Baboons
In 12 untreated control animals, 111In-platelets
accumulated rapidly on segments of vascular graft, reaching plateau
values by 75 minutes (Fig 2), with propagated
fibrin-rich thrombotic tails developing after 30 minutes and also
peaking by 75 minutes (Fig 3). By imaging
analysis, neither platelet-rich vascular graft thrombus nor
fibrin-rich propagated tails showed significant decrement during the
subsequent 60 minutes of concurrent heparin administration (Figs 2 and 3).
|
, results in untreated controls. Right, Fibrin
accumulation; open bar, controls. Injections of r-LysPgn at 4 and 8
mg/kg reduce platelet deposition on segments of vascular graft but
do not decrease fibrin accumulation in vascular graft thrombi.
|
Substantial amounts of fibrin accumulated after placement of
thrombogenic segments of vascular grafts in both vascular graft thrombi
(2.9±0.8 mg; Fig 2
) and propagated thrombotic tails (6.9±2.3 mg; Fig 3).
During initial thrombus formation (0 to 75 minutes), thrombin
production was greatly amplified, as shown by the 10-fold
increase in plasma concentrations of TAT complex (Table 1) and the
4-fold elevation in the plasma levels of FPA, the thrombin cleavage
product of fibrinogen (Table 1).
Effects of r-LysPgn on Thrombus Formation on Vascular
Grafts
111In-platelet deposition on segments of vascular
graft was reduced by prethrombosis injections of r-LysPgn in a
dose-dependent manner (Fig 2). For 8-mg/kg dosing, platelet
deposition on segments of vascular graft was reduced to
1.5±0.5x109 platelets from 3.4±1.4x109
platelets in untreated controls (P=.011 by ANOVA). The
4-mg/kg dose of r-LysPgn also decreased platelet deposition
significantly in vascular graft thrombus (P=.036 by ANOVA).
However, the 125I-fibrin content of vascular graft thrombi
did not correlate with injected doses of r-LysPgn (Fig 2). Thus,
elevated levels of plasminogen resulting from injections of
r-LysPgn produced reciprocal decreases in platelet deposition (Figs 2 and 3 and Table 1).
111In-platelet deposition was also significantly
decreased in the propagated thrombotic tails at 135 minutes after the
8-mg/kg dose of r-LysPgn before thrombosis (Fig 3; P=.016 by
ANOVA). However, the accumulation of 125I-fibrin in the
propagated thrombotic tails was not altered by bolus injections of
r-LysPgn (Fig 3; P>.1 in all cases).
Increases in thrombin production (TAT) and thrombin cleavage
products (FPA) in plasma during thrombus formation remained
unaltered by bolus injections of r-LysPgn (Table 1). Plasma
D-dimer levels were also unchanged by prethrombosis
injections of 8 mg/kg r-LysPgn (Table 1).
Template BT determinations were not affected by any of the doses of
r-LysPgn administered (Fig 4).
|
In vitro platelet aggregation induced by ADP, collagen, or
TRAP1-6 was impaired by r-LysPgn (Table 2).
The in vitro addition of r-LysPgn (125 µg/mL) to PRP in amounts
approximately equivalent to physiological levels of
native Glu-plasminogen impaired the responsiveness of the
platelets to aggregatory agonists, as shown by the increase in
agonist concentration required to induce half-maximal aggregatory
responses (AC50), ie, from 3.4±1.3 to 5.5±2.1
µmol/L for ADP (P<.05), from 0.77±0.54 to 1.64±0.70
µg/mL for collagen (P<.05), and from 11.0±4.0 to
15.0±4.6 µmol/L for TRAP (P<.05), after r-LysPgn
preincubation, compared with control plasma. However, when PRP was
prepared from animals before and after they received bolus r-LysPgn (8
mg/kg), no impairment in platelet aggregatory responsiveness to
physiological agonists was detectable ex vivo
(Table 2; P>.3).
|
Effects of r-LysPgn on Stent Thrombosis
In a separate group of 5 animals, 111In-platelet
deposition, 125I-fibrin accumulation, and changes in
thrombosis blood tests were measured for metallic endovascular stents
deployed in long-term exteriorized AV shunts beginning 30 minutes after
injection of r-LysPgn (8 mg/kg IV) and continuing for 60 minutes.
Platelet deposition on stents was reduced by r-LysPgn from
2.43±1.15x109 to 0.95±0.55x109 (Fig 5;
P=.0003 by ANOVA).
|
Effects of r-LysPgn on TPA-Dependent Thrombolysis
The infusion of TPA (0.2 mg/kg) beginning at 75 minutes after
thrombus was established and continuing for 60 minutes (concurrent with
the infusion of heparin) was subthreshold in that there was no
significant decrease in 111In-platelet deposition in
vascular graft thrombi (Fig 6) or in propagated
thrombotic tails (Fig 7). Similarly,
125I-fibrin accumulation in vascular graft thrombi (Fig 6)
was not changed by 0.2 mg/kg TPA. These data are in accord with
previous observations in the same model system.23
|
). Effects of
subthreshold-dose TPA (0.1 mg/kg) with low-dose r-LysPgn (2 mg/kg)
administered before thrombosis (
) or after thrombosis (
)
demonstrate significant reduction in platelet deposition by low
doses of r-LysPgn and subthreshold-dose TPA when thrombus forms in the
presence of r-LysPgn. Fibrin accumulation is not affected by these
regimens (right).
|
By contrast, 111In-platelet deposition was
substantially reduced in vascular graft thrombi when low-dose bolus
r-LysPgn (2 mg/kg) was injected before thrombosis (before the
thrombogenic segments of vascular graft were placed in the AV shunts)
and was followed by the infusion of a subthreshold dose of TPA (0.1
mg/kg) for 60 minutes beginning after thrombosis, ie, at 75 minutes
(Fig 6; P=.011 by ANOVA). The accumulation of
125I-fibrin was significantly decreased in propagated
thrombotic tails (Fig 7; P=.05 by ANOVA). The combination of
low-dose r-LysPgn and subthreshold-dose TPA produced significant
prolongation of template BT measurements, ie, 7±3 versus 3.5±1
minutes in controls (P=.02; Fig 4).
When bolus low-dose r-LysPgn (2 mg/kg) was injected after thrombosis at
75 minutes with subthreshold-dose TPA (0.1 mg/kg) infusions,
111In-platelet deposition in vascular graft thrombi did
not significantly decrease (Fig 6; P>.3), and the
deposition on the propagated thrombotic tails was similar to
prethrombosis injections of r-LysPgn (Fig 7; P>.5).
125I-fibrin accumulation was not decreased in vascular
graft thrombi (Fig 6; P>.5) or propagated thrombotic tails
(Fig 7; P>.1) by postthrombosis low-dose r-LysPgn. However,
postthrombosis injections of r-LysPgn significantly prolonged
the BT, ie, 11±4 versus 3.0±0.3 minutes in controls
(P=.006; Fig 4).
Thrombin production was not significantly altered by
subthreshold-dose TPA administration (Table 1; TAT levels
P>.2 in all cases and FPA levels P>.2 in all
cases). However, plasma D-dimer levels appeared to be
significantly increased with prethrombosis low-dose r-LysPgn (2 mg/kg)
and subsequent infusion of subthreshold-dose TPA (0.1 mg/kg) (Table 1;
P=.05).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study demonstrates in baboon models of thrombus formation and dissolution that r-LysPgn produces dose-dependent reduction in stent and vascular graft thrombosis by greatly enhancing thrombolysis without detectable effects on hemostasis. These findings predict that clinical thrombosis will be significantly decreased at sites of interventional vascular procedures without impairing hemostasis by injection of r-LysPgn before invasive elective procedures are performed.
The clinical relevance of the results in this report is strengthened by particular features of the models used in the study. First, the thrombus to be lysed is preformed in vivo from autologous constituents and incorporates radiolabeled autologous platelets and homologous fibrinogen. This design obviates the limitations imposed by use of blood clots produced in vitro with heterologous components. Second, both arterial-type platelet-rich and venous-type fibrin-rich thrombi are formed separately, thereby permitting independent analyses regarding the relative responsiveness of platelet-rich versus fibrin-rich thrombi in assessing different thrombolytic therapies. Third, the use of 111In-platelets and noninvasive gamma camera imaging provides continuous measurements of platelet deposition in real time, yielding independent time-course analysis of the relative thrombolytic responsiveness for platelet-rich vascular graft thrombus versus fibrin-rich propagated tail thrombus. Fourth, by use of nonhuman primates, recombinant human molecules are readily evaluated directly, and immunological reagents specific for human antigens may be used in measuring primate-specific immunometric blood tests of thrombosis, fibrinolysis, and hemostasis. Fifth, because the measurements are quantitative and reproducible, important intermediate outcomes can be compared efficiently with fewer animals. Finally, because flow through the thrombotic device is maintained throughout each study, the thrombotic:thrombolytic interface remains efficient and steady state, rendering the evaluation more sensitive to significant changes in thrombogenesis and thrombolysis. The rationale for adding heparin in this model to stabilize nonoccluding thrombus after 75 minutes is based on observations in nonheparinized animals showing consistent thrombo-occlusion by 100±15 minutes, frequently preceded by irregular fragmentary embolic losses.23 Thus, heparin interrupts thrombus propagation produced by soluble thrombin without blocking thrombogenesis mediated by bound thrombin.44 45
The mechanism(s) underlying the antithrombotic effects of r-LysPgn are
attributable to enhanced thrombolysis. The amount of
thrombus accumulating at sites of forming thrombus represents
the net balance between active processes promoting thrombus
accumulation and active processes dissipating thrombus. The present
data show that r-LysPgn greatly amplifies endogenous
TPA-dependent thrombolysis, thereby augmenting thrombus
dissipation, resulting in less accumulated thrombus. This conclusion is
strongly supported by the observation that the combination of
subthreshold bolus r-LysPgn (2 mg/kg) and less-than-subthreshold bolus
TPA (0.1 mg/kg) act synergistically to markedly decrease thrombus
formation (Fig 6; P=.011).
Electron microscopic studies demonstrate that Lys-plasminogen is preferentially incorporated into the fibrin structure and modulates the fibrin network.46 Lys-plasminogen appears to facilitate end-to-end and center-to-end binding to fibrin monomers and fragment X.46 Lys-plasminogen also enhances the rate at which fibrin forms and produces altered fibrin structure having increased optical density compared with the fibrin clot formed in the presence of Glu-plasminogen.47 48 This increase in clot turbidity produced by Lys-plasminogen is related to enhanced lateral association of fibrin protofibrils and consequent greater fiber thickness,49 50 giving rise to a coarse and loose fibrin gel structure with increased fluid space. Lys-plasminogen binds to fibrin with 10-fold-greater affinity than Glu-plasminogen,51 and the affinity increases further when fibrin is partially degraded by plasmin.52 The resultant fibrin is more easily solubilized.47 Thus, prethrombosis administration of r-LysPgn in the baboon model appears to promote the formation of fibrin-fibrin structure enriched with prothrombolytic r-LysPgn and highly susceptible to TPA-dependent lysis. Because activated coherent platelets are incorporated in thrombus by fibrin bridging, modified fibrin-fibrin structure might also explain the selective reduction in platelet recruitment without any decrease in fibrin accumulation into forming thrombus that was observed experimentally. Altered fibrin-fibrin coupling may also explain the observed reduction in fibrin accumulation and the enhanced TPA lysis observed by Mehta et al,21 in dogs as well as the augmented solubility observed in vitro.47 53 54 This interpretation is in accord with the evidence in this report that r-LysPgn must be incorporated into the forming thrombus to exhibit its prothrombolytic effects. These data also imply that clinical application will be limited to elective procedures.
The significance of r-LysPgn–dependent impairment in platelet aggregatory responsiveness in vitro is less certain,55 56 57 58 because the inhibitory effects of r-LysPgn on platelet aggregation are not observed ex vivo after the injection of r-LysPgn into animals, and no impairment was observed in platelet hemostatic function, such as prolongation in the template BT. Conversely, the inhibitory effects of r-LysPgn on platelet deposition are much more selective than on fibrin accumulation, implying some platelet-inhibitory mechanism.
Thus, in this baboon model of thrombus formation and dissolution, the inclusion of r-LysPgn into forming thrombus decreases the amount of accumulating thrombus by greatly enhancing fibrinolysis and with little impairment in hemostatic function. We postulate that injection of r-LysPgn before therapeutic interventional procedures are performed will significantly reduce thrombosis and complicating thrombo-occlusive episodes without impairing hemostasis. Thus, r-LysPgn may find useful application in patients undergoing angioplasty and stent deployment.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported in part by research grants from the National Institutes of Health HL-41619, HL-43667, and RR-00165. We wish to thank Gregory Annisette, Evan Dessasau, Deborah White, Elke Gottfriedsen, and Jeanette Lundquist for expert technical assistance.
Received September 11, 1996; revision received February 13, 1997; accepted February 13, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Fuster V, Stein B, Ambrose JA, Badimon L, Badimon JJ, Chesebro JH. Atherosclerotic plaque rupture and thrombosis: evolving concepts. Circulation. 1990;82(suppl II):II-47-II-59.
2. Maseri A. Pathogenic components of acute ischemic syndromes. Circulation. 1990;81:1-3.
3. Willerson JT, Golino P, Eidt J, Campbell WB, Buja LM. Specific platelet mediators and unstable coronary artery lesions: experimental evidence and potential clinical implications. Circulation. 1989;80:199-205.
4. Coller BS. Platelets and thrombolytic therapy. N Engl J Med. 1990;322:33-42.
5. AIMS Trial Study Group. Effect of intravenous APSAC on mortality after acute myocardial infarction: preliminary report of a placebo-controlled clinical trial. Lancet. 1988;1:545-549.
6. GISSI (Gruppo Italiano per lo Studio della Streptochinasi nell' infarto miocardico). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet. 1986;1:397-402.
7. ISIS Steering Committee. Intravenous streptokinase given within 0-4 hours of onset of myocardial infarction reduced mortality in ISIS-2. Lancet. 1987;1:502-510.
8. Chesebro JH, Knatterud G, Braunwald E. Thrombolytic therapy. N Engl J Med. 1988;319:1544-1545.
9. TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial: phase I findings. N Engl J Med. 1985;312:932-936.
10. Topol EJ, Califf RM, George BS, Kereiakes DJ, Abbottsmith CW, Candela RJ, Lee KL, Pitt B, Stack RS, O'Neill WW. A randomized trial of immediate versus delayed elective angioplasty after intravenous tissue plasminogen activator in acute myocardial infarction. N Engl J Med. 1987;317:581-588.
11. Lijnen HR, Nelles L, Van Hoef B, deCock F, Collen D. Biochemical and functional characterization of human tissue-type plasminogen activator variants obtained by deletion and/or duplication of structural/functional domains. J Biol Chem. 1990;265:5677-5683.
12. Madison EL, Goldsmith EJ, Gerard RD, Gething MJ, Sambrook JF. Serpin-resistant mutants of human tissue-type plasminogen activator. Nature. 1989;339:721-724.
13. Jang I, Gold HK, Leinbach RC, McNary JE, Fallon JT, Collen D. Acceleration of reperfusion by combination of rt-PA and a selective thrombin inhibitor, argatroban. Circulation. 1989;80(suppl II):II-217. Abstract.
14. Rudd MA, George D, Johnstone MT, Moore RT, Collins L, Rabbani LE, Loscalzo J. Effect of thrombin inhibition on the dynamics of thrombolysis and on platelet function during thrombolytic therapy. Circ Res. 1992;70:829-834.
15. Yasuda T, Gold HK, Leinbach RC, Yaoita H, Fallon JT, Guerrero L, Napier MA, Bunting S, Collen D. Kistrin, a polypeptide platelet GPIIb/IIIa receptor antagonist, enhances and sustains coronary arterial thrombolysis with recombinant tissue-type plasminogen activator in a canine preparation. Circulation. 1991;83:1038-1047.
16. Collen D, Lijnen HR, Verstraete M. Fibrinolytic system and its disorders. In: Handin RI, Lux SE, Stossel TP, eds. Blood: Principles and Practice of Hematology. Philadelphia, Pa: JB Lippincott; 1995:1261-1288.
17. Suenson E, Thorsen S. The course and prerequisites for Lys-plasminogen formation during fibrinolysis. Biochemistry. 1988;27:2435-2443.
18. Fredenburg JC, Nesheim ME. Lys-plasminogen is a significant intermediate in the activation of Glu-plasminogen during fibrinolysis in vitro. J Biol Chem. 1992;267:26150-26159.
19. Badylak SF, Voytik SL, Henkin J, Burke S, Sasahara AA, Simmons A. The beneficial effect of Lys-plasminogen upon the thrombolytic efficacy of urokinase in a dog model of peripheral arterial thrombosis. Haemostasis. 1991;21:278-285.
20. Chen LY, Mehta JL. Lys- and glu-plasminogen potentiate the inhibitory effect of recombinant tissue plasminogen activator on human platelet aggregation. Thromb Res. 1994;74:555-563.
21. Mehta JL, Chen L, Nichols WW, Johannesen M, Bregengard C, Hedner U, Saldeen TG. Recombinant lys-plasminogen, but not glu-plasminogen, improves recombinant tissue-type plasminogen activator-induced coronary thrombolysis in dogs. J Am Coll Cardiol. 1995;25:753-760.
22. Busby SJ, Mulvihill E, Rao D. Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity. J Biol Chem. 1991;266:15286-15292.
23. Runge MS, Harker LA, Bode C, Ruef J, Kelly AB, Marzec UM, Caban R, Shaw S-Y, Haber E, Hanson SR. Enhanced thrombolytic and antithrombotic potency of a fibrin-targeted plasminogen activator in baboons. Circulation. 1996;94:1412-1422.
24. Savage B, McFadden PR, Hanson SR, Harker LA. The relation of platelet density to platelet age: survival of low and high-density 111Indium-labeled platelets in baboons. Blood. 1986;68:386-393.
25. Harker LA, Hanson SR, Kirkman TR. Experimental arterial thromboembolism in baboons: mechanism, quantitation, and pharmacologic prevention. J Clin Invest. 1979;64:559-569.
26. Hanson SR, Harker LA. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone. Proc Natl Acad Sci U S A. 1988;85:3184-3188.
27. Kelly AB, Marzec UM, Krupski W, Bass A, Cadroy Y, Hanson SR, Harker LA. Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood. 1991;77:1006-1012.
28. Hanson SR, Kotze HF, Savage B, Harker LA. Platelet interactions with Dacron vascular grafts: a model of acute thrombosis in baboons. Arteriosclerosis. 1985;5:595-603.
29. Gruber A, Hanson SR, Kelly AB, Yan BS, Bang N, Griffin JH, Harker LA. Inhibition of thrombus formation by activated recombinant protein C in a primate model of arterial thrombosis. Circulation. 1990;82:578-585.
30. Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970;170:1095-1096.
31. Harker LA, Kelly AB, Hanson SR. Experimental arterial thrombosis in non-human primates. Circulation. 1991;83(suppl IV):IV-41-IV-55.
32. Hanson SR, Powell JS, Dodson T, Lumsden A, Kelly AB, Anderson JS, Clowes AW, Harker LA. Effects of angiotensin converting enzyme inhibition with cilazapril on intimal hyperplasia in injured arteries and vascular grafts in the baboon. Hypertension. 1991;18(suppl II):II-70-II-76.
33. Krupski WC, Bass A, Kelly AB, Hanson SR, Harker LA. Reduction in thrombus formation by placement of endovascular stents at endarterectomy sites in baboon carotid arteries. Circulation. 1991;84:1749-1757.
34. Lumsden AB, Kelly AB, Schneider PA, Krupski WC, Dodson T, Hanson SR, Harker LA. Lasting safe interruption of endarterectomy thrombosis by transiently infused antithrombin peptide D-Phe-Pro-ArgCH2Cl in baboons. Blood. 1993;81:1762-1770.
35. Hanson SR, Pareti FI, Ruggeri ZM, Kunicki TJ, Montgomery RR, Zimmerman TS, Harker LA. Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon. J Clin Invest. 1988;81:149-158.
36. Hanson SR, Harker LA, Bjornsson TD. Effects of platelet-modifying drugs on arterial thromboembolism in baboons: aspirin potentiates the antithrombotic actions of dipyridamole and sulfinpyrazone by mechanism(s) independent of platelet cyclooxygenase inhibition. J Clin Invest. 1985;75:1591-1599.
37. Harker LA, Kelly AB, Hanson SR, Krupski W, Bass A, Osterud B, FitzGerald GA, Goodnight SH, Connor WE. Interruption of vascular thrombus formation and vascular lesion formation by dietary n-3 fatty acids in fish oil in nonhuman primates. Circulation. 1993;87:1017-1029.
38. Cadroy Y, Hanson SR, Kelly AB, Marzec UM, Evatt BL, Kunicki TJ, Montgomery RR, Harker LA. Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in non-human primates. Blood. 1994;83:3218-3224.
39. Hanson SR, Wilcox JN, Kelly AB, Lumsden AB, Scott NA, Harker LA. Thrombin mediation of arterial smooth muscle cell (SMC) proliferation after mechanical vascular injury in non-human primates. Blood. 1992;80(suppl I):166a. Abstract.
40. Kelly AB, Hanson SR, Dunwiddie CT, Harker LA. Safe lasting interruption of arterial thrombosis by transient intravenous administration of tick anticoagulant peptide (TAP). Circulation. 1992;86(suppl I):I-411. Abstract.
41. Harker LA, Slichter SJ. The bleeding time as a screening test for evaluation of platelet function. N Engl J Med. 1972;287:155-159.
42. Harker LA, Malpass TW, Branson HE, Hessel I, Slichter SJ. Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet dysfunction associated with selective alpha granule release. Blood. 1980;56:824-834.
43. Malpass TW, Hanson SR, Savage B, Hessel EA, Harker LA. Prevention of acquired transient defect in platelet plug formation by infused prostacyclin. Blood. 1981;57:736-740.
44. Cadroy Y, Hanson SR, Harker LA. Antithrombotic effects of synthetic pentasaccharide with high affinity for plasma antithrombin III in non-human primates. Thromb Haemost. 1993;70:631-635.
45. Yokoyama T, Kelly AB, Marzec UM, Hanson SR, Kunitada S, Harker LA. Antithrombotic effects of orally active synthetic antagonist of activated factor X in nonhuman primates. Circulation. 1995;92:485-491.
46. Weisel JW, Nagaswami C, Korsholm B, Petersen LC, Suenson E. Interactions of plasminogen with polymerizing fibrin and its derivatives, monitored with a photoaffinity cross-linker and electron microscopy. J Mol Biol. 1994;235:1117-1135.
47. Petersen LC, Suenson E. Effect of plasminogen and tissue-type plasminogen activator on fibrin gel structure. Fibrinolysis. 1991;5:51-59.
48. Garman AM, Smith RAG. The binding of plasminogen to fibrin: evidence for plasminogen-bridging. Thromb Res. 1982;27:311-320.
49. Carr ME, Shen LL, Hermans J. Mass-length ratio of fibrin filters from gel permeation and light scattering. Biopolymers. 1977;16:1-15.
50. Carr ME, Hermans J. Size and density of fibrin fibers from turbidity. Macromolecules. 1978;11:46-50.
51. Suenson E, Thorsen S. Secondary site binding of glu-plasminogen, lys-plasminogen and miniplasmin to fibrin. Biochem J. 1981;197:619-628.
52. Suenson E, Lutzen O, Thorsen S. Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis. Eur J Biochem. 1984;140:513-522.
53. Carr ME, Gabriel DA. The effect of dextran 70 on the structure of plasma-derived fibrin gels. J Lab Clin Med. 1980;96:985-993.
54. Aberg M, Hedner U, Bergentz S-E. The antithrombotic effect of dextran. Scand J Haemost. 1979;34(suppl):61-68.
55. Aronson DL, Chang P, Kessler CM. Platelet-dependent thrombin generation after in vitro fibrinolytic treatment. Circulation. 1992;85:1706-1712.
56. Niewiarowski S, Senyi AF, Gilles P. Plasmin induced platelet aggregation and platelet release reaction. J Clin Invest. 1973;52:1647-1659.
57. Lu H, Soria C, Li H, Soria J, Lijnen HR, Perrot JY, Caen JP. Role of active center and lysine binding sites of plasmin in plasmin-induced platelet activation and disaggregation. Thromb Haemost. 1991;65:67-72.
58. Stricker RB, Wong D, Shiu DT, Reyes PT, Sherman M. Activation of plasminogen by tissue plasminogen activator on normal and thrombasthenic platelets: effects on surface proteins and platelet aggregation. Blood. 1986;68:275-280.
This article has been cited by other articles:
 |
|
 |
|
  E. I. Tucker, U. M. Marzec, T. C. White, S. Hurst, S. Rugonyi, O. J. T. McCarty, D. Gailani, A. Gruber, and S. R. Hanson Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI Blood, January 22, 2009; 113(4): 936 - 944. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||