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(Circulation. 1995;93:792-799.)
© 1995 American Heart Association, Inc.

Articles

Does Antithrombotic Therapy Influence Residual Thrombus After Thrombolysis of Platelet-Rich Thrombus?

Effects of Recombinant Hirudin, Heparin, or Aspirin

Presented in part at the 63rd Scientific Sessions of the American Heart Association, Dallas, Tex, November 12-15, 1990; 40th Annual Scientific Session, American College of Cardiology, Atlanta, Ga, March 3-7, 1991; and 41st Annual Scientific Session, American College of Cardiology, Dallas, Tex, April 12-16, 1992.

Jozef S. Mruk, MD, PhD; Pierre Zoldhelyi, MD; Mark W.I. Webster, MB, CHB; Magda Heras, MD; Diane E. Grill, MS; David R. Holmes, Jr, MD; Valentin Fuster, MD; James H. Chesebro, MD

From the Division of Cardiovascular Diseases and Internal Medicine (J.S.M., P.Z., M.W.I.W., M.H., D.E.G., D.R.H. Jr., J.H.C.), Mayo Clinic and Mayo Foundation, Rochester, Minn; and Mount Sinai Medical Center and School of Medicine (V.F.), New York, NY.

Correspondence to James H. Chesebro, MD, Cardiovascular Institute, Box 1030, Mount Sinai Medical Center, One Gustave L. Levy Pl, New York, NY 10029-6574.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Thrombolysis to normal flow in patients with acute myocardial infarction preserves left ventricular function and decreases mortality. Failure of early reperfusion, reocclusion, or residual thrombus may be due to concurrent activation of the platelet-coagulation system. Thus, we hypothesized that the best concomitant antithrombotic therapy (recombinant [r]-hirudin, heparin, or aspirin) will maximally accelerate thrombolysis by r–tissue-type plasminogen activator (rTPA) and reduce residual thrombus.

Methods and Results Occlusive thrombi were formed in the carotid arteries of 29 pigs (by balloon dilatation followed by endarterectomy at the site of injury-induced vasospasm) and matured for 30 minutes before rTPA was started, with or without antithrombotic therapy. Thrombolysis was assessed with the use of angiography and measurement of residual thrombus. Pigs were allocated to one of five treatments: placebo, rTPA, rTPA plus r-hirudin, rTPA plus heparin, or rTPA plus intravenous aspirin. No placebo-treated pig reperfused. Two of six animals treated with rTPA alone reperfused compared with seven of seven animals treated with rTPA plus r-hirudin (reperfusion time, 33±10 minutes), six of seven animals treated with rTPA plus heparin (reperfusion time, 110±31 minutes), and two of six animals with rTPA plus aspirin. The activated partial thromboplastin time was prolonged in only the rTPA plus r-hirudin group (25±0.1 times baseline) and the rTPA plus heparin group (5.3±0.2 times baseline). Residual ¹¹¹In-platelet and ¹²⁵I-fibrin(ogen) depositions were lower in the heparin-treated group and lowest in the r-hirudin–treated group (heparin versus hirudin, respectively; incidence of residual macroscopic thrombus was six of six animals versus two of seven [P=.01]; ¹²⁵I-fibrin(ogen), 170±76 versus 48±6 x10⁶ molecules/cm² [P=.02]; ¹¹¹In-platelets, 47±15 versus 13±2 x10⁶/cm², P=.10). No pigs developed spontaneous bleeding.

Conclusions Thrombin inhibition with heparin or r-hirudin significantly accelerated thrombolysis of occlusive platelet-rich thrombosis, but only the best antithrombotic therapy (r-hirudin) eliminated or nearly eliminated residual thrombus.

Key Words: thrombosis • platelets • fibrin

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Prompt treatment of evolving myocardial infarction with thrombolytic agents improves survival and preserves cardiac function.^{1 2 3 4} Despite the use of heparin and aspirin, however, recanalization of the occluded coronary artery (which predicts myocardial salvage) is achieved in only 50% to 80% of patients, depending in part on the thrombolytic agent that is used.^{1 4 5 6 7} Complete reperfusion (TIMI grade 3 flow) at 90 minutes improves 24-hour and 30-day mortality and myocardial function but is achieved in only 29% to 54% of patients.⁴ Simultaneous ongoing thrombosis appears to limit thrombolysis.^{8 9 10 11} Because residual thrombus is more thrombogenic than deeply injured artery,¹² therapy should target its dissolution.

Activation of coagulation during pharmacological thrombolysis activates platelets, generates thrombin, occurs with both SK and rTPA, and may be mediated by plasmin or SK or directly by rTPA.^{8 9 10} Thrombin generation during thrombolysis may reflect paradoxical activation of prothrombin,^{8 13} exposure of coagulantly active thrombin bound to fibrin,^{14 15 16 17} or both. The rate and extent of thrombolysis are limited, probably by thrombin-related mechanisms.^{11 18 19}

New, specific direct thrombin inhibitors have been more effective than heparin in preventing platelet-dependent thrombosis after arterial injury.^{20 21 22 23 24 25} Unlike heparin, these agents react directly with thrombin to form enzyme/inhibitor complexes.^{19 20 24}

Because thrombosis and lysis are dynamic and simultaneous processes,^{11 26} we hypothesized that the best antithrombotic therapy (r-hirudin) at the dose that prevented macroscopic mural thrombus after deep injury^{21 22} would maximally increase the rate and extent of thrombolysis compared with aspirin, placebo, or heparin. We used the dose of heparin that had a measurable aPTT, reduced mural thrombus, and had previously enhanced thrombolysis in dogs.^{20 21 26} This dose (200 U/kg over the first hour) is higher than conventional doses of heparin used in coronary care units. Extensive deep arterial injury, with platelet-rich thrombotic occlusion, was created with balloon angioplasty and directional endarterectomy. Major end points were incidence of residual thrombus, quantitative residual ¹¹¹In-platelet and ¹²⁵I-fibrin(ogen) depositions, and incidence of and time of reperfusion.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Twenty-nine 4-month-old pigs (mean weight, 35.0±0.4 kg) of Babcock four-way cross stock (a mixture of Landrace, Yorkshire, Hampshire, and Durock breeds) were assigned to treatment with either placebo (three pigs), rTPA (six pigs), rTPA and r-hirudin (seven pigs), rTPA and heparin (seven pigs), or rTPA and aspirin (six pigs). Drug administration during the experiments was not blinded, but all subsequent data and tissue and sample analyses were performed without knowledge of the treatment administered. The study was approved by the Mayo Clinic Animal Care Committee and conformed with American Heart Association guidelines.

Iodination of Porcine Fibrinogen
Porcine fibrinogen (Sigma Chemical Co) was dissolved in buffer containing 150 mmol/L NaCl, 5 mmol/L trisodium citrate, and 20 mmol/L Tris-HCl, pH 7.4, and was centrifuged at 12 000g for 10 minutes at room temperature to remove insoluble particles. Next, the fibrinogen solution was treated with 5 mmol/L diisopropyl fluorophosphate for 2 hours at 4°C and dialyzed three times for 4 hours at 4°C against 100-fold excess of the buffer. After final dialysis, fibrinogen preparation was again centrifuged at room temperature for 10 minutes at 12 000g. Fibrinogen preparations were more than 92% clottable with thrombin in vitro. After iodination in batches with ¹²⁵I, fibrinogen solution was stored in aliquots at -70°C until use in each porcine experiment.

Porcine fibrinogen was iodinated with ¹²⁵I.^{27 28} More than 93% of the radioactivity of labeled fibrinogen was perceptible with tricholoroacetic acid. Coomassie blue staining and autoradiography of sodium dodecyl sulfate/polyacrylamide gel electrophoresis of iodinated fibrinogen showed a single band on unreduced gels and three bands on the reduced gels. Iodinated fibrinogen was more than 95% clottable with thrombin in vitro.

Platelet Labeling With ¹¹¹In-Tropolone
Autologous femoral arterial blood (43 mL) was drawn into 7 mL of adenosine/citrate/dextrose anticoagulant and platelets labeled with ¹¹¹In-tropolone.^{22 28}

Experimental Protocol
One day before surgery, autologous ¹¹¹In-labeled platelets (300 µCi) and ¹²⁵I-fibrinogen (250 µCi) were (re)injected into the pigs. On the next day, pigs were sedated with 1000 mg ketamine IM (Ketaset, Bristol Laboratories), intubated, and mechanically ventilated with room air (Harvard respirator, Harvard Apparatus). Anesthesia was maintained with an infusion of etomidate (40 mg/L; Abbott Laboratories), fentanyl (10 mg/L; Abbott), and ketamine (1000 mg/L) at 3 to 5 mL/min. The ECGs and intra-arterial pressures were monitored continuously.

Formation of Carotid Occlusive Thrombus
After general anesthesia and intubation, bilateral femoral cutdowns were performed to expose both femoral arteries and veins. Cannulation was performed in the right femoral artery for continuous arterial pressure monitoring, the right femoral vein for venous blood sampling, the left femoral vein for infusion of drugs, and the left femoral artery for angioplasty catheter access. An 0.8x3-cm balloon dilation catheter (Blue Max, Medi-tech) was advanced to the proximal left carotid artery under fluoroscopic guidance. A standardized dilation protocol was performed of five inflations to 6 atm for 30 seconds, with 60 seconds between inflations. We have previously shown that deep arterial injury (tear through the internal elastic lamina into the media) is created by this protocol in 70% to 80% of dilated arteries.^{21 22 29} Platelet-rich mural thrombi and, occasionally, occlusive thrombi form over areas of deep arterial injury. Severe vasospasm always occurs immediately distal to the balloon-induced deep injury site.^{29 30} The area of vasospasm (approximately 5 cm from the bifurcation of the carotid arteries) was identified, marked on the skin with metallic clips, and used to create a localized consistent area of deep arterial injury, with increased flow, velocity, and shear rate. An 11F guiding sheath, with side holes to maintain blood flow, was placed in the proximal left carotid artery, and Simpson's directional endarterectomy device (AtheroCath, Devices for Vascular Intervention) was positioned at the level of the distal vasospasm. Six longitudinal endarterectomy cuts (approximately 1.5x9 mm), with 60° rotation between each pass, resulted in consistent, localized injury in the media. Occlusive platelet-rich thrombi formed within minutes over the area of endarterectomy.

Before arterial injury, each pig received a bolus of 25 U/kg heparin to prevent clotting on the catheters and stasis thrombosis caused by the 11F guiding sheath. The biological half-life of this dose of heparin in pigs is approximately 12 minutes, and platelet deposition at the site of deep arterial injury is not significantly affected.^{21 31} After endarterectomy, the Simpson's device and guiding sheath were withdrawn. The thrombus was allowed to mature for 30 minutes, and a 7F right Judkins diagnostic angiographic catheter (Cordis) with a metallic ring marker for calibration was placed just distal to the carotid bifurcation. To exclude occlusion of the left carotid artery secondary to vasospasm, nitroglycerin (300 µg over 1 minute) was injected into the proximal left carotid artery at the end of the thrombus maturation period. Lack of blood flow by contrast angiography after nitroglycerin injection indicated that the filling defect was caused by occlusive thrombus and not by vasospasm.

Time to Reperfusion
Carotid angiography was performed with 6 mL of ionic contrast (Renographin 76, Squibb). Spot films were taken before angioplasty, immediately after angioplasty, after 30 minutes of thrombus maturation, and after nitroglycerin administration. During the subsequent infusions of thrombolytic regimens (or placebo), angiograms of the left carotid artery were performed every 10 minutes for the first 90 minutes and then every 30 minutes for the next 120 minutes. The study protocol is summarized in Fig 1.

View larger version (17K): [in this window] [in a new window]   Figure 1. Experimental design for formation of occlusive thrombus in porcine carotid artery, timing for administration of treatment, and timing and contrast angiographic observations (arrows).

Treatment Administration
R-Alteplase (single-chain rTPA, Genetech, Inc) was given as a bolus of 0.3 mg/kg, followed by a continuous infusion of 2 mg/kg per hour for the first 90 minutes and then at 0.5 mg/kg per hour for the next 120 minutes. On the basis of previous dose-ranging studies,²² r-desulfato-hirudin (CGP 39393, CIBA-GEIGY Pharmaceuticals) was administered at the lowest infusion dose that prevented thrombus (bolus, 1 mg/kg; infusion, 0.7 mg/kg per hour for 210 minutes). On the basis of dose-ranging study of six heparin doses,²¹ heparin from porcine intestine (Elkins-Sinn, Inc) was administered at the lowest dose that reduced platelet-rich thrombus (bolus, 100 U/kg; infusion, 100 U/kg per hour for 210 minutes). Heparin or r-hirudin was started at the same time as rTPA. L-Lysine acetylsalicylic acid (Bayer) equivalent to 5 mg/kg of acetylsalicylic acid (aspirin) was administered as an intravenous bolus 15 minutes before rTPA infusion. This dose of aspirin completely inhibited aggregation of porcine platelets induced by arachidonic acid in whole blood.^{32 33}

Tissue Preparation and Analysis
At 210 minutes after the start of treatment, 120 mL of filtered 0.5% Evans blue dye solution (Sigma) in 0.9% saline was injected into the descending aorta to demarcate the extent of arterial injury. After the pigs were killed with an overdose of pentobarbital, the proximal descending aorta was immediately cannulated and the carotid arteries were perfused with buffered 0.9% saline until the effluent from external jugular vein incisions was clear. The vessels were then perfused with buffered 2% glutaraldehyde for 15 minutes. All perfusions were done at physiological pressure. After fixation in situ, the carotid arteries were harvested and cleaned of adventitia. The area of injury of the left carotid artery was documented by sketches on a 1:1 scale and cut into 1-cm segments for subsequent analysis: isotope counting and macroscopic and microscopic examinations. Platelet and fibrin(ogen) depositions on arterial segments were quantified according to the method of Dewanjee.²⁸ Counting for ¹¹¹In was performed on the day of surgery and, for ¹²⁵I, 3 weeks later, as previously described.^{21 22} In segments positive for Evans blue dye staining or thrombus, at least two histological sections per segment were stained with van Gieson's elastic and hematoxylin and eosin stains and examined with light microscopy for deep injury. Each artery was examined for macroscopic thrombus with a 2x magnifier as previously described.^{21 22 29}

Hemostatic Parameters
Blood samples for hemostatic measurements (anticoagulated with 0.13 mol/L trisodium citrate; ratio of anticoagulant to blood, 1:10) were drawn via two-syringe technique before and 90 minutes after the start of treatment and immediately before the animals were killed at 210 minutes. Platelet counts, hematocrits, and aPTTs were determined with the use of standard methods as previously described.^{21 22} Samples for fibrinogen levels were supplemented with aprotinin (200 IU/mL blood, Sigma).^{34 35} Plasma samples for fibrinogen determination were stored at -70°C. Plasma fibrinogen was measured in batches as described by Jacobsson.³⁶ Thrombin-clottable plasma protein (fibrinogen) was determined after collection of clots on wooden applicator sticks and subsequent solubilization, using the biuret method (Total Protein Kit, Sigma).^{37 38}

Fibrinolytic Activity of rTPA, UK, and SK Against Human and Porcine Plasminogen-Rich Fibrin
Fibrinolytic activity was measured according to the fibrin plate lysis method as described by Haverkate and Brakman.³⁹ Drops (35 µl) of each concentration of fibrinolytic agents were applied, and fibrin plates were incubated for 18 hours at 37°C at 100% relative humidity.^{39 40 41} rTPA (it was assumed that 2 ng rTPA=1 IU)^{42 43} was obtained from Genetech, Inc; UK (Abbokinase) was from Abbott Laboratories; and SK (Streptase) was from Hoechst-Russel Pharmaceuticals, Inc. Fibrinolytic reagents were dissolved as recommended by the manufacturers, divided into small aliquots, and stored at -70°C. For experiments, stock solutions of fibrinolytic reagents were diluted to the desired concentrations with gelatin buffer.³⁹ Human plasminogen-rich fibrinogen (Grade-L) was obtained from Kabi-Vitrum Inc, and bovine thrombin (Thrombinar) was obtained from Jones Medical Industries. Porcine plasminogen-rich fibrinogen was obtained from Sigma Chemical Co, and all other reagents were from Sigma or Fisher Scientific Co and were the highest available quality.

Plasminogen content in porcine and human fibrinogens was determined by incubating solutions of fibrinogens with 1000 U/mL UK for 2 hours at 37°C to allow activation of plasminogen to plasmin. Standard curves were constructed activating the porcine and human plasminogens (Sigma) under the same conditions. Plasmin activities were determined with the chromogenic substrate Spectrozyme PL (American Diagnostica Inc). Plasminogen content of porcine fibrinogen (42.5 µg/mg fibrinogen) was similar to human fibrinogen (32 µg/mg fibrinogen).

Statistical Analysis
Results are presented as mean±SEM. The three animals in the placebo group were not included in any analyses. Residual platelets and molecules of fibrin(ogen) per centimeter squared of artery were analyzed after a logarithmic transformation was applied to normalize the data. One-way ANOVA was used to test for any differences between all of the treatment groups in residual platelets, molecules of fibrin(ogen) per cm² of artery, hematocrit, platelet counts, and fibrinogen. When the one-way ANOVA was statistically significant (P<.05), the Student's t test was used for pairwise group comparisons. Because the time until reperfusion did not have a normal distribution, a Kruskal-Wallis test was used to test for an overall group difference. The Wilcoxon rank-sum test was then used to compare groups.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Fibrinolytic Activity of rTPA, UK, and SK Against Human and Porcine Plasminogen-Fibrin In Vitro
Dosing of rTPA was based on in vitro determinations of fibrinolytic activity (Fig 2). SK did not activate porcine plasminogen. Porcine plasminogen was approximately 10-fold more resistant to activation by rTPA than was human plasminogen, necessitating higher doses of rTPA for porcine than for human studies.

View larger version (20K): [in this window] [in a new window]   Figure 2. Fibrinolytic activity in vitro of rTPA (), UK (), and SK () against human (open symbols) and porcine (closed symbols) plasminogen-rich fibrin. (Note: SK does not activate porcine plasminogen.72 )

Outcome and Reperfusion
Times to reperfusion are summarized in Fig 3. No animal reperfused in the placebo group. Of six animals treated with rTPA alone, one reperfused at 90 minutes and one reperfused at 120 minutes (mean, 105 minutes). Macroscopic and microscopic examinations of the injured carotid arteries confirmed the presence of a platelet-rich occlusive thrombi overlying an area of deep arterial injury. Typically, platelet thrombi were coated with a fibrin- and erythrocyte-rich layer. The pig that reperfused at 90 minuets died suddenly at 120 minutes. Necropsy did not show hemorrhagic or embolic complications.

View larger version (39K): [in this window] [in a new window]   Figure 3. Time to reperfusion of occluded carotid arteries in control and treated groups. Each horizontal bar represents one pig with occlusion (shaded) and patency (open) indicated over time. n indicates number of animals. Mean aPTT ratios to control ±SEM are listed for each group.

In the rTPA-plus-heparin group, six of seven pigs reperfused (at 10, 70, 70, 120, 180, and 210 minutes; mean, 110 minutes). After reperfusing at 70 minutes, one pig reoccluded and developed a large perivascular hematoma of the neck at 120 minutes, secondary to carotid artery perforation, resulting in airway compromise and termination of the study at 180 minutes. The second pig that reperfused at 70 minutes died suddenly at 150 minutes. No hemorrhagic or embolic complications were found on necropsy.

Of seven animals treated with rTPA and r-hirudin, all reperfused (at 10, 10, 30, 40, 40, 50, and 50 minutes; mean, 33 minutes), and two transiently reoccluded but reopened. None had hemorrhagic complications during the 210 minutes of study.

Reperfusion occurred in only two of six pigs receiving adjunctive aspirin treatment with rTPA: one at 60 minutes and the other at 80 minutes. The former animal died suddenly at 180 minutes (unremarkable necropsy); the latter reoccluded at 150 minutes and did not reperfuse again.

Residual Thrombus, Platelets, and Fibrin(ogen)
Fig 4A and 4B summarizes platelet and fibrin(ogen) deposition in the left carotid arteries. Residual ¹¹¹In-platelet deposition was 355±106, 123±23, 13±2, 47±15, and 328±96 x10^-6/cm² with placebo, rTPA alone, rTPA plus hirudin, rTPA plus heparin, and rTPA plus aspirin, respectively. Residual ¹²⁵I-fibrin(ogen) deposition x10¹² molecules/cm² was 1712±503, 714±261, 48±6, 170±76, and 1389±246 in the same respective groups as ¹¹¹In-platelet deposition. Statistical differences between groups are summarized in Fig 4A and 4B.

View larger version (40K): [in this window] [in a new window]   Figure 4. A, Residual 111In-platelet deposition per cm2 at the end of 210 minutes of treatment. Bar indicates the mean value; vertical line, SEM; and n, number of pigs in each treatment group. B, Residual 125I-fibrin(ogen) deposition per cm2 at the end of 210 minutes of treatment. Bar indicates mean value; vertical line, SEM; and n, number of pigs in each treatment group.

In the rTPA group, both of the animals that reperfused had marked mural thrombus at euthanasia. In the rTPA-plus-aspirin group, both of the two reperfused pigs had extensive mural thrombi. All six reperfused pigs receiving rTPA plus heparin had residual thrombi (marked in five and minimal in one) at necropsy. In contrast, no thrombus was found in five pigs, and only minimal residual macroscopic mural thrombus was found in two of seven reperfused pigs receiving rTPA plus r-hirudin (P=.01 versus heparin). Representative angiograms of residual arterial obstruction in pigs receiving adjuvant therapy are shown in Fig 5 (note improved patency after administration of rTPA plus r-hirudin).

View larger version (127K): [in this window] [in a new window]   Figure 5. Representative angiograms of left carotid artery in pigs treated with rTPA alone (left), rTPA plus heparin (middle), and rTPA plus r-hirudin (right). Note long segments with extensive residual irregular thrombus in both non–hirudin-treated pigs and clear lumen in hirudin-treated pigs.

Hemostatic Parameters
In all groups, the hematocrit and platelet count decreased slightly but not significantly at 210 minutes or death. The aPTT remained normal throughout the experiments in the control, rTPA, and rTPA-plus-aspirin groups. In the rTPA–plus–r-hirudin group, the aPTT was prolonged to 2.5±0.1 times baseline control value at 90 minutes and to 2.7±0.2 times baseline control at 210 minutes. In the rTPA-plus-heparin group, the aPTT was prolonged to 5.3±0.6 and 7.3±0.5 times baseline control at 90 minutes and at 210 minutes, or death, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This is the first report of quantitative measurement of residual thrombus burden after thrombolysis. Direct thrombin inhibition with r-hirudin, compared with heparin, significantly accelerated the rate and extent of lysis of occlusive platelet-rich thrombi by rTPA after deep arterial injury in the pig; r-hirudin eliminated residual thrombus in five of seven pigs compared with none of six pigs receiving heparin (P=.01). Fibrin(ogen) deposition was also greater in heparin- than in hirudin-treated pigs (P=.02). r-Hirudin was used at an aPTT of only 2.5 times control (clinically acceptable with lytic agent) compared with heparin at an aPTT of 5.3 times control (clinically unacceptable with lytic agent). aPTTs have a unique relation to the blood level of each thrombin inhibitor and do not indicate the antithrombotic efficacy.⁴⁴ However, high aPTTs (>3 times control) in humans receiving lytic therapy are associated with an increased incidence of intracerebral bleeds.^{45 46} Both heparin and r-hirudin significantly decreased residual platelet and fibrin(ogen) deposition after lysis compared with aspirin or rTPA alone. In all seven pigs receiving rTPA plus r-hirudin, all carotid arteries were open by 50 minutes, and all remained widely patent at 210 minutes, with only minimal residual thrombus in two.

No spontaneous bleeding was observed in any of the pigs. Dosing of heparin and hirudin was based on prior dose-ranging studies to find the lowest dose reducing or eliminating thrombus.^{21 22} The bolus dose rapidly increased the aPTT and blood level to plateau but not above^{21 22} to minimize the risk for bleeding. In human studies in which bleeding resulted with r-hirudin and thrombolysis, the aPTT after the bolus dose significantly exceeded the subsequent plateau level.^{45 46}

The increased rate and extent of lysis for normal early reflow (TIMI grade 3 flow) appear to be critical for reduced mortality and improved myocardial function.⁴ Because rehospitalization for angina, reinfarction, and revascularization over the next year occurs in more than 50% of patients,⁴⁷ reduced thrombus burden (shown to be feasible in this study) in patients may reduce these clinical events.

Thrombin is pivotal for the formation, growth, maintenance, and consolidation of thrombus. Direct inhibition with r-hirudin (a specific probe for thrombin), but not heparin, blocks these processes and leads to dissolution of thrombus.^{12 20 21 22 48 49} Reasons for the powerful protective effect of hirudin over heparin may relate to active thrombin binding to fibrin and arterial wall substrates, which causes conformational changes in thrombin and poor binding by heparin/antithrombin III but no change in binding by hirudin.^{17 50} In addition, platelet secretion and fibrin formation produce natural inhibitors of heparin action but no inhibitors against hirudin.^{51 52 53 54} Substantial amounts of thrombin remain bound to fibrin in thrombus.^{14 15 16 17} Heparin is 20 to 50 times less effective than r-hirudin in inhibiting thrombin bound to fibrin compared with thrombin in solution.¹⁷ This probably occurs because receptors on fibrin-bound thrombin are masked for binding to heparin/antithrombin III and heparin/heparin cofactor II. In addition, platelet factor 4 (released from platelets) and generated fibrin monomer II interfere with heparin and heparin/antithrombin III action.^{50 51 52 53 54} Antithrombins with greater binding affinity to thrombin have greater antiplatelet potential and at lower anticoagulant dosage (lower aPTT). Graded reduction in thrombin affinity of r-hirudin mutants resulted in a progressive attenuation of antiplatelet activity with no change in inhibition of fibrin formation and was not reflected by measurement of the aPTT.⁵⁵ Thrombin inhibitors with lower binding affinities have not prevented mural thrombus in deeply injured arteries, even at high doses that result in very prolonged aPTTs.⁵⁵

Clinical data indicate that after lytic therapy for acute myocardial infarction, concomitant aspirin plus heparin therapy to an aPTT of 60 to 85 seconds is associated with very low reocclusion rates (4.9% to 6.4%) 1 week after thrombolysis⁴ and is dependent on the level of anticoagulation.^{18 56 57 58} The pilot study of r-hirudin versus heparin for thrombolysis in acute myocardial infarction supports the superiority of r-hirudin over heparin plus aspirin in improving early reflow and reducing reocclusion, ischemic pain, reinfarction, or death.⁵⁶ The median delay of 40 minutes in starting randomized therapy with r-hirudin or heparin after thrombolytic therapy in TIMI 9 requires analysis and may account for no difference between therapies.⁵⁹

Aspirin did not reduce the time to reperfusion or platelet or fibrin(ogen) deposition compared with rTPA alone. With thrombus as a substrate for thrombosis, aspirin alone or added to heparin did not reduce growth of thrombus.¹² Asprin has a borderline effect on rethrombosis after thrombolysis in dogs.^{9 60} Aspirin dramatically improves the overall outcome of thrombolytic therapy in large clinical trials.^{2 18} Decreased reocclusion probably plays a major role because aspirin does not appear to accelerate thrombolysis, time to reperfusion after deep arterial injury, or platelet-rich arterial thrombosis, as suggested by a meta-analysis and shown by this study.⁶¹ The central core of the thrombus contains much less stainable thrombin than thrombus adjacent to deeply injured artery.⁶² This may explain the reduction in reocclusion but not mural thrombus by aspirin.

Heparin at the dosage used in this study reduced the incidence of mural thrombus to 25% after deep injury compared with 76% with placebo in the prior dose-ranging studies.^{21 22} Cercek et al²⁶ first reported the acceleration of arterial thrombolysis with rTPA by pretreating dogs with 200 U/kg heparin. Acceleration of thrombolysis by heparin in dogs was later seen with pro-urokinase,⁶³ SK, and UK.⁶⁴ Choice of a fibrin-rich arterial thrombus model (coronary copper coil) and choice of a species that responds to low levels of plasminogen activators (dog)²⁶ make comparison with our study difficult. Disrupted human plaques produce large platelet-rich thrombi at the injury site and fibrin-erythrocyte–rich distal or proximal extensions.^{65 66 67} Platelet-rich thrombi are relatively resistant to plasminogen activators^{68 69} and also require higher levels of hirudin for prevention.^{61 70} Thus, dose-response depends in part on type of injury, type of thrombus, and species.

The effects of heparin at this dose on other coagulation proteases, such as factor Xa, are of less physiological importance than thrombin.^{71 72} In addition, administration of rTPA with lower-dose r-hirudin (aPTT of 1.5 to 2.0 times control) in dogs with coronary thrombosis induced by electrical (mild) injury accelerated lysis and prevented reocclusion, whereas heparin at doses with similarly low aPTTs did not accelerate lysis and prevented reocclusion.⁶⁰

In summary, the best antithrombotic therapy (inhibition of thrombin by r-hirudin) not only accelerates thrombolysis but also eliminates or reduces residual platelet-rich thrombus after deep arterial injury. The high dose of heparin required for reduction of mural thrombus and acceleration of thrombolysis produces an excessive prolongation of the aPTT (5 to 6 times control) for routine clinical use. However, direct thrombin inhibition by r-hirudin at a dose that prolonged the aPTT to a clinically relevant 2.5 times control accelerated thrombolysis with rTPA, almost completely eliminated residual thrombus, and appears promising for treatment in humans.

	Selected Abbreviations and Acronyms

 

aPTT = activated partial thromboplastin time r = recombinant SK = streptokinase TPA = tissue-type plasminogen activator UK = urokinase

View this table: [in this window] [in a new window]   Table 1. Platelet and Fibrin(ogen) Deposition

	Acknowledgments

 
Dr Mruk was the recipient of a fellowship from NIH (HL-07111/14T); Dr Webster was the recipient of a fellowship from the National Heart Foundation of New Zealand and the Fogarty Center, NIH; Dr Heras was the recipient of a research grant from CIRIT, Generalitat de Catalunya, Spain; and Dr Chesebro was funded in part by NIH grant HL-39959.

Received November 9, 1994; revision received August 24, 1995; accepted September 25, 1995.

	References

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