Pierre Zoldhelyi

Recombinant hirudin in patients with chronic, stable coronary artery disease. Safety, half-life, and effect on coagulation parameters.

BackgroundBecause the specific antithrombin hirudin prevents platelet-rich arterial thrombus and accelerates thrombolysis in a variety of animal models, it has promise as antithrombotic therapy. We therefore studied the half-life, effect on anticoagulant parameters, and safety of hirudin in patients with coronary artery disease. Methods and ResultsThirty-eight men and 1 woman (age [mean±SD], 60.4±6.9 years) with angiographic coronary disease were allocated in a single-blind ascending dosage study to a 6-hour IV infusion of recombinant hirudin (CGP 39 393) or matching placebo. The median terminal half-life for hirudin, measured by ELISA, was 2.7, 2.3, 2.9, 3.1, and 2.0 hours for the 0.02, 0.05, 0.1, 0.2, and 0.3 mg · kg−1 · h−1 groups, respectively. Activated partial thromboplastin times (aPTT) at 3, 4, and 6 hours were averaged into a plateau value. The aPTT plateau-to-baseline ratios were 1.5±0.1, 2.0±0.1, 2.3±0.1, 2.7±0.1, and 2.9±0.1, respectively, with hirudin infused at 0.02, 0.05, 0.1, 0.2, and 0.3 mg · kg−1 h−1. From 62% to 77% of the aPTT plateau value was seen within 30 minutes of starting the infusions and was directly related to dose. The aPTT-to-baseline ratios correlated well with plasma hirudin levels (r=.88), whereas poor correlation and sensitivity were observed between plasma hirudin levels and activated coagulation time (ACT)-to-baseline ratios (r=.44). Plasma levels of hirudin and ACT in seconds correlated overall well (r=.80), but considerable overlap occurred between baseline ACT and ACT at plasma hirudin concentrations <1000 ng/mL. Prothrombin times were significantly prolonged only at a dosage of 20.05 mg · kg−1 · h−1 and were 11.8±0.5 (INR=1.0), 12.3±0.7 (INR=1.1), 13.3±1.2 (INR=1.4), 14.2±0.4 (INR=1.7), and 15.8±0.9 (INR=2.3) seconds for each respective hirudin dosage. Thrombin times were beyond range (>600 seconds) at 6 hours in all except 2 patients who received the lowest dosage. All parameters returned to baseline between 8 and 18 hours after the infusion. Bleeding times were not significantly prolonged. No side effects occurred. No antibodies to hirudin were detected 2 weeks after the infusion. ConclusionsRecombinant hirudin has a terminal half-life of 2 to 3 hours. The aPTT correlates well with plasma levels of hirudin and allows close titration over a wide range of anticoagulation, while ACT and prothrombin time are relatively insensitive for monitoring hirudin administration. At anticoagulant levels effective in experimental thrombosis, a 6-hour infusion of hirudin is well tolerated and safe in a predominantly male group of patients with stable coronary atherosclerosis.

Persistent thrombin generation in humans during specific thrombin inhibition with hirudin.

BackgroundThe degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. Methods and ResultsIntravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg−1·h−1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin- hirudin complex increased from 794 ± 85 pg/mL (mean ± SEM) 15 minutes after the start of the infusion to 1617 ± 151 pg/mL at 6 hours of infusion to 2667 ± 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 ± 0.40 at baseline to 1.64 ± 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 ± 0.87 at 24 hours (nonsignificant compared with baseline). ConclusionsMeasurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin- hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.

Pathogenesis of thrombosis in unstable angina

Plaque rupture of the thinned, weak fibrous cap infiltrated by macrophages and overlying a pool of lipid in the arterial wall initiates the acute thrombotic event of unstable angina. Thrombosis may be advanced within minutes. Most lesions that precede plaque rupture are minor (less than 50% stenosis); thus, thrombus greatly contributes to sudden flow limitation and onset of symptoms. If thrombosis can be totally blocked (not possible with current antithrombotic agents), clinical events should be preventable, and endogenous thrombolysis may be possible within days. Local and systemic factors contribute to arterial thrombosis. With type III injury (fissure into plaque or media) platelet-rich thrombus anchors in the fissure, tracks along the site of deep injury, extends into the lumen, and requires the highest blood level of specific thrombin inhibition (a molar concentration that inhibits the total concentration of prothrombin in circulating blood). Thus, the thrombin content requiring inhibition in type III injury is highest. Local factors for thrombosis associated with type III injury include the rheology of blood flow (increased shear rate forces platelets to the periphery) and substrates in the arterial wall. Plaque substrates include the more thrombogenic collagens (types I and III and diabetic or glycosylated collagen), tissue thromboplastin, lipid gruel, thrombin bound to arterial wall matrix, and decreased prostacyclin. There is a direct relation between platelet deposition (thrombus) and local vasoconstriction, which may perpetuate each other. Thrombus as a substrate is more thrombogenic than type III arterial injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Does Antithrombotic Therapy Influence Residual Thrombus After Thrombolysis of Platelet-Rich Thrombus? Effects of Recombinant Hirudin, Heparin, or Aspirin

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 x 10(6) molecules/cm2 [P = .02]; 111In-platelets, 47 +/- 15 versus 13 +/- 2 x 10(6)/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.

Adenoviral Gene Transfer of Fortilin Attenuates Neointima Formation Through Suppression of Vascular Smooth Muscle Cell Proliferation and Migration

Background—Fortilin, a recently characterized nuclear antiapoptotic factor structurally distinct from inhibitor of apoptosis proteins (IAPs) and Bcl-2 family member proteins, has been suggested to be involved in cell survival and regulation of apoptosis within the cardiovascular system. In this continued investigation, we characterized the influence of adenovirus-mediated fortilin (Ad-fortilin) gene delivery on vascular remodeling after experimental angioplasty. Methods and Results—Vessel wall expression of Ad-fortilin or adenoviral luciferase (Ad-luc) was demonstrated 72 hours and 14 days after rat carotid artery (CA) balloon angioplasty. Morphometric analyses 14 days after injury revealed significantly diminished neointima development in the Ad-fortilin–treated CAs compared with Ad-luc or PBS controls, with no changes in medial wall morphometry observed between the 3 groups. The Ad-fortilin–treated CAs demonstrated a 50% reduction in medial wall proliferating cell nuclear antigen (PCNA) labeling after 72 hours, with significantly reduced neointimal and medial wall PCNA labeling and cell counts after 14 days. Terminal dUTP nick-end labeling results and morphological changes characteristic of programmed cell death suggest a trend toward reduced apoptosis in the fortilin-transfected balloon-injured vessels compared with Ad-luc injured controls. Temporal analysis of human aorta smooth muscle cell (SMC) proliferation demonstrated a marked time-dependent inhibition in Ad-fortilin treated SMCs without the influence of elevated apoptosis. Thymidine incorporation was significantly inhibited in the Ad-fortilin–treated cells compared with Ad-luc controls. Ad-fortilin transfected SMCs also demonstrated significantly decreased migration compared with Ad-luc controls. Conclusions—These cumulative results suggest that the novel antiapoptotic protein fortilin may play important redundant pathophysiological roles in modulating the vascular response to experimental angioplasty through suppression of SMC proliferation and migration concomitant with reduction of vessel wall apoptosis.

Plaque disruption and thrombosis in unstable angina pectoris

Therapeutic strategies and clinical trials in unstable angina should be based on the pathogenesis, risk, and mechanisms of thrombosis. The mechanisms of thrombosis and the differences in the effects of anticoagulant, antithrombotic, antifibrin, and antiplatelet drugs must be taken into account when determining the dosage and duration of therapy. Ignoring these principles may prevent identification of new therapy, increase the cost of new drug development and research, increase the cost of new drugs, and increase the cost of medical care.

Growth Suppression of Human Coronary Vascular Smooth Muscle Cells by Gene Transfer of the Transcription Factor E2F-1

BackgroundThe transcription factor E2F-1 promotes S-phase entry and death in transformed cells and primary cardiomyocytes. We tested the hypothesis that overexpression of E2F-1 forces growth-arrested human coronary vascular smooth muscle cells (VSMCs) to enter the S phase, undergo apoptosis, and thereby regulate VSMC growth. Methods and ResultsEarly-passage (≤5 passages) coronary VSMCs were transduced at an MOI of 100 with a recombinant adenovirus encoding human E2F-1. E2F-1 expression was observed by immunohistochemistry as early as 6 to 8 hours after exposure of the VSMCs to Ad.E2F-1 but not to the control vector Ad.RR. When cells were kept in growth-arrest medium, 40% of Ad.E2F-1–treated VSMCs entered the S phase by 96 hours, whereas the percentage remained <5% in Ad.RR-treated cells. Transition to the S phase in the E2F-1–transduced VSMCs was followed by apoptosis, as reflected by chromatin condensation, membrane blebbing, cell detachment, and loss of mitochondrial membrane integrity. E2F-1 overexpression resulted in positive dUTP nick end-labeling mediated by terminal deoxynucleotidyl transferase, associated with a robust increase in caspase 3–like activity. Four days after infection with Ad.E2F-1, the fraction of hypodiploid VSMCs in subG1 increased to 75%. At 7 days, gene transfer of E2F-1 had completely suppressed the growth of VSMCs, whereas the number of Ad.RR-infected cells had increased >8 times. ConclusionsOverexpression of the transcription factor E2F-1 regulates growth of human coronary VSMCs by forcing the cells to enter the S phase and then to die. Cell death appears to involve caspase 3–like activity, which, in the VSMCs, is markedly increased by overexpression of E2F-1.