Reduction in Stent and Vascular Graft Thrombosis and Enhancement of Thrombolysis by Recombinant Lys-Plasminogen in Nonhuman Primates
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.
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
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 β-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 (8×10 pixels) and separately for the fibrin-rich propagated tail for 20 cm immediately distal to the vascular graft (80×10 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
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 300×103/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
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.
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⇓).
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⇓).
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±0.5×109 platelets from 3.4±1.4×109 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±1.15×109 to 0.95±0.55×109 (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
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).
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
|BPTI||=||bovine pancreas trypsin inhibitor|
|TAT||=||thrombin–antithrombin III complex|
|TPA||=||tissue plasminogen activator|
|TRAP||=||thrombin receptor agonist peptide|
|UPA||=||urokinase plasminogen activator|
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.
- Copyright © 1997 by American Heart Association
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