Alessandra Mailhac

Characterization of the relative thrombogenicity of atherosclerotic plaque components: Implications for consequences of plaque rupture☆

OBJECTIVES The purpose of this study was to determine whether different components of human atherosclerotic plaques exposed to flowing blood resulted in different degrees of thrombus formation. BACKGROUND It is likely that the nature of the substrate exposed after spontaneous or angioplasty-induced plaque rupture is one factor determining whether an unstable plaque proceeds rapidly to an occlusive thrombus or persists as a nonocclusive mural thrombus. Although observational data show that plaque rupture is a potent stimulus for thrombosis, and exposed collagen is suggested to have a predominant role in thrombosis, the relative thrombogenicity of different components of human atherosclerotic plaques is not well established. METHODS We investigated thrombus formation on foam cell-rich matrix (obtained from fatty streaks), collagen-rich matrix (from sclerotic plaques), collagen-poor matrix without cholesterol crystals (from fibrolipid plaques), atheromatous core with abundant cholesterol crystals (from atheromatous plaques) and segments of normal intima derived from human aortas at necropsy. Specimens were mounted in a tubular chamber placed within an ex vivo extracorporeal perfusion system and exposed to heparinized porcine blood (mean [+/- SEM] activated partial thromboplastin time ratio 1.5 +/- 0.04) for 5 min under high shear rate conditions (1,690 s-1). Thrombus was quantitated by measurement of indium-labeled platelets and morphometric analysis. Under similar conditions, substrates were perfused with heparinized human blood (2 IU/ml) in an in vitro system, and thrombus formation was similarly evaluated. RESULTS Thrombus formation on atheromatous core was up to sixfold greater than that on other substrates, including collagen-rich matrix (p = 0.0001) in both heterologous and homologous systems. Although the atheromatous core had a more irregular exposed surface and thrombus formation tended to increase with increasing roughness, the atheromatous core remained the most thrombogenic substrate when the substrates were normalized by the degree of irregularity as defined by the roughness index (p = 0.002). CONCLUSIONS The atheromatous core is the most thrombogenic component of human atherosclerotic plaques. Therefore, plaques with a large atheromatous core content are at high risk of leading to acute coronary syndromes after spontaneous or mechanically induced rupture because of the increased thrombogenicity of their content.

Effect of an eccentric severe stenosis on fibrin(ogen) deposition on severely damaged vessel wall in arterial thrombosis. Relative contribution of fibrin(ogen) and platelets.

BackgroundCoronary thrombosis is a dynamic process dependent on the pathological substrate, the local shear forces, and blood factors. Methods and ResultsWe investigated the effect of a severe (80%) eccentric stenosis on fibrin(ogen) interaction with a deeply damaged vessel wall, its relation to platelet deposition in thrombus formation, and the influence of time on thrombus growth. Porcine 125I-fibrinogen and autologous 111In-platelets were injected into pigs instrumented for extracorporeal circulation and treated with low-dose heparin (aPTT ratio < 1.5) that has been previously shown and herein confirmed not to affect platelet and/or fibrin(ogen) attachment. Tunica media, as a model of severely injured vessel wall, was mounted in a tubular perfusion chamber containing an eccentric axisymmetric sinusoidal stenosis obstructing the lumen and exposed for 1, 5, and 10 minutes to perfusing blood. A shear rate of 424 s−1 at the laminar, parallel parabolic local flow perfused segments one to two orders of magnitude greater at the apex of the stenosis. Fibrin(ogen) deposition, its axial distribution with respect to the apex, and its relation to platelet deposition were determined by an ex vivo analysis of the test substrates. Fibrin(ogen) and platelet deposition were both significantly higher at the apex of the stenosis than at either the prestenotic or poststenotic area at all the studied perfusion times (P<.02). However, fibrin(ogen) deposition demonstrated a significantly smaller degree of increase from the prestenotic area to the apex as well as a smaller degree of decrease from the latter to the poststenotic region, compared with platelet deposition (P<.05). Although both fibrin(ogen) and platelet deposition increased over time, the ratio of fibrin(ogen) to platelets showed a progressive decrease that became significant from 5 to 10 minutes (P<.03) at either low or high shear rate. The rate of platelet deposition was relatively constant; however, fibrin(ogen) deposition progressively decreased, especially at the apex. ConclusionsOn severely damaged vessel wall, fibrin(ogen) and platelet deposition is maximal at the apex of the stenosis where shear rate is extremely high and parallel streamlines are deformed. Nevertheless, fibrin(ogen) deposition is significantly less dependent on high shear rate than is platelet deposition, and the pattern is not influenced by time. Finally, fibrin(ogen) deposition appears to be predominant in the thrombus layers adjacent to a severely damaged vessel wall regardless of the local shear stress levels and flow conditions.

A new approach for local intravascular drug delivery. Iontophoretic balloon.

BACKGROUND Catheter-based systems are being developed to deliver drugs directly into the vessel wall. Pressure-mediated trauma and lack of homogeneous delivery are key limitations of these approaches. METHODS AND RESULTS We studied a new catheter-based delivery system that uses electrical current to force the drug into the vessel wall. The in vivo feasibility of this approach has been assessed by delivering 125I-hirudin into porcine carotid arteries. Vascular levels of hirudin after active iontophoresis (4 mA/cm2, 5 minutes) were 80-fold greater than those achieved by passive diffusion (without electricity). Tissue hirudin levels declined over time; by 1 hour after delivery, 80% of the drug had left the vessel wall, and by 3 hours later, the levels of hirudin within the wall were similar to those achieved by passive diffusion. Autoradiography revealed distribution of the drug throughout the entire circumference of the arterial wall within the intima, media, and adventitia. Iontophoresis-mediated vessel wall trauma was minimal (less than 10% endothelial denudation and medial smooth muscle cell damage). Balloon injury after local delivery changed neither kinetics nor distribution of the drug into the arterial wall. CONCLUSIONS (1) High local concentrations of hirudin in the arterial wall may be achieved with the iontophoretic balloon catheter. (2) The drug is distributed throughout the entire vessel wall without significant damage. (3) The retention of hirudin in the arterial wall is time dependent. (4) This technique might be useful to deliver therapeutic agents before or after percutaneous vascular interventions.

Local delivery of r-hirudin by a double-balloon perfusion catheter prevents mural thrombosis and minimizes platelet deposition after angioplasty.

BACKGROUND The major morbidity of percutaneous transluminal coronary angioplasty is acute thrombosis and restenosis of the dilated lesion. Platelet-thrombus deposition occurs within minutes after injury, is primarily mediated by thrombin, causes acute occlusion, and contributes to late restenosis. Experimentally, specific thrombin inhibitors have prevented mural thrombosis. However, local therapy may be more effective than systemic treatment. We tested the hypothesis that high local concentrations of an antithrombin drug at the site of arterial injury following balloon angioplasty inhibit platelet thrombus formation equally or better than conventional systemic treatment and at lower systemic anticoagulant levels. METHODS AND RESULTS Balloon angioplasty of the carotid arteries of 29 pigs was performed using systemic intravenous treatment with heparin (100 U/kg, groups I and II), suboptimal r-hirudin (0.3 mg/kg, group III), and higher-dose r-hirudin (0.7 mg/kg, group IV), which is the lowest dose that completely inhibited arterial thrombosis in the pig. Immediately after balloon angioplasty of the first carotid, additional local therapy with placebo (group I) or r-hirudin (groups II, III, and IV; 0.3 mg/kg in 1 mL) was administered with distal perfusion through a new percutaneous double-balloon catheter. After 1 hour of local drug delivery, angioplasty of the contralateral carotid was performed. Reflow for 1 hour was permitted to both carotids to compare the short-term effect of local plus systemic treatment with systemic treatment on quantitative 111In-labeled platelet deposition and macroscopic mural thrombus formation on deeply injured carotid segments. Local drug delivery of placebo compared with systemic heparin treatment resulted in no change of platelet deposition (x 10(6)/cm2, mean +/- SEM) in controls (group I, 91.0 +/- 23.5 versus 80.8 +/- 19.4), but local delivery of r-hirudin resulted in a significant reduction in group II (15 +/- 2.5 versus 71.3 +/- 14.5; P < .02) and group III (11.4 +/- 2.5 versus 80.5 +/- 11.4; P < .01) and was borderline in group IV (7.4 +/- 1.8 versus 14.1 +/- 7.4; P = .05), respectively. The incidence of macroscopic mural thrombus formation with local and systemic treatment was 86% and 75% in group I, 16% and 70% in group II, 14% and 71% in group III, and 0% and 16% in group IV, respectively. CONCLUSIONS Local therapy with the specific thrombin inhibitor r-hirudin significantly reduces short-term quantitative platelet deposition and macroscopic mural thrombus formation following balloon angioplasty compared with systemic treatment of conventional doses of heparin and hirudin and requires a significantly smaller amount of the recombinant drug.

Inhibition of growth of thrombus on fresh mural thrombus. Targeting optimal therapy.

BACKGROUND Residual mural thrombus on severely damaged arterial wall is very thrombogenic. We tested the hypothesis that direct thrombin inhibition will block thrombus growth on fresh thrombus better than indirect thrombin inhibition, cyclooxygenase inhibition, or both. METHODS AND RESULTS A fresh mural thrombus was formed by directly perfusing fresh porcine blood for 5 minutes over severely damaged arterial wall at a high shear rate in a well-characterized ex vivo perfusion system. The average platelet (P) and fibrinogen (F) deposition (D) achieved in 5 minutes were 382 +/- 32 x 10(6) platelets/cm2 and 296 +/- 36 x 10(12) fibrinogen molecules/cm2, respectively. Thrombus growth on the fresh mural thrombus was quantitated by directly perfusing blood from pigs with 111In-labeled platelets and 125I-labeled fibrinogen for an additional 5 minutes over the preformed mural thrombus. Treatment included recombinant hirudin (1 mg/kg per hour IV) as a probe for thrombin, aspirin (5 mg/kg IV) as a platelet inhibitor of cyclooxygenase, heparin (moderate, 100 IU/kg per hour IV; high-dose, 250 IU/kg per hour IV) as an indirect thrombin inhibitor, and heparin (100 IU/kg per hour) plus aspirin (5 mg/kg IV). Thrombus growth as measured by labeled PD (x 10(6)/cm2) and FD (x 10(12) molecules/cm2) was mildly but not significantly reduced by aspirin (1034 +/- 92 and 436 +/- 78, respectively) compared with baseline (1113 +/- 67 and 545 +/- 52, respectively). Inhibition of thrombus growth with heparin was dose dependent. A regression analysis showed an inverse correlation of PD and FD with mean plasma heparin concentrations (r = -.81, P = .0001 and r = -.49, P = .0007, respectively). Recombinant hirudin led to a profound inhibition of thrombus growth (PD, 30 +/- 12; FD, 109 +/- 21), which was significant compared with all groups, even the highest dosage of heparin (250 IU/kg per hour). CONCLUSIONS Specific thrombin inhibition markedly inhibits platelet and fibrinogen deposition onto fresh mural thrombus at a high shear rate. Aspirin alone or in combination with heparin has little effect on evolving thrombosis. Heparin dose dependently reduces thrombus growth, but even the highest dosage is less effective than hirudin. Thrombin appears to be the primary activator of platelets by fresh thrombus.

Different response to balloon angioplasty of carotid and coronary arteries : effects on acute platelet deposition and intimal thickening

PTCA is a well-established intervention to reduce the severity of atherosclerotic coronary stenosis. Its primary success rate is seriously handicapped by the high incidence of late restenosis. Given the clinical and social importance of this proliferative process, new strategies are needed to prevent or reduce restenosis. Several animal models as well as different arteries have been used to study neointimal proliferation after arterial injury. A number of agents have shown to reduce neointimal proliferation after arterial injury in the carotids and iliac arteries of rodent models. Unfortunately, these results have not been replicated in humans. We have compared the acute and late response to vascular injury of the carotid and coronary arteries in the pig. Arterial injury was induced by performing balloon angioplasty of the carotid (elastic) and coronary (muscular) arteries in swine. Acute platelet-thrombus formation was evaluated by quantitation of Indium-labeled platelets deposited on the injured segments 1 h after procedure. Measurement of intimal area was performed by morphometry of the most stenotic cross-section at 28 days after balloon angioplasty. Platelet deposition after mild and severe injury in carotids (4 +/- 1 and 56 +/- 13 x 10(6) platelets/cm2, respectively) and coronaries (15 +/- 5 and 141 +/- 20 x 10(6) platelets/cm2, respectively) are significantly greater in deep, than in mild injury (P < 0.005), and significantly greater in coronary than in carotid arteries after deep injury (P < 0.05). Likewise, late neointima formation was significantly greater (P < 0.05) after mild and severe injury in coronary (17 +/- 0.5 and 56 +/- 2%, respectively) than in carotid arteries (5 +/- 0.5 and 12 +/- 1%, respectively). Acute platelet-thrombus formation and late neointimal thickening are modulated by the degree of injury induced during the interventions; and after disruption of the internal elastic lamina, coronary arteries always had significantly more acute thrombus and neointimal thickening. This study emphasizes the importance of the animal species, the type of injury and the artery chosen for studies on restenosis post interventions.

Short- and Long-Term Evolution of Unstented Nonocclusive Coronary Dissection After Coronary Angioplasty

OBJECTIVES We assessed the short- and long-term clinical and angiographic outcome of nonocclusive unstented dissection after percutaneous transluminal coronary angioplasty (PTCA) and its correlation with restenosis. BACKGROUND The use of stents has dramatically increased both the number and the cost of coronary revascularization procedures. However, this technique is not completely risk free, and its benefits have not been fully demonstrated in uncomplicated dissections. METHODS We studied 129 consecutive patients with 49 nonocclusive dissections after PTCA (grades A to D of National Heart, Lung, and Blood Institute classification) and good distal flow (TIMI [Thrombolysis in Myocardial Infarction] flow grade 3). All patients underwent coronary angiography at 24 h and at six months post-PTCA. Clinical status was assessed every three months in the outpatient clinic. Study subjects were matched with 60 other patients in whom stenting was performed for the presence of dissection. RESULTS In the former group, all but two patients (with type E dissection, which evolved to coronary occlusion and myocardial infarction) improved their dissection score during follow-up: at six months only 18 dissections were still angiographically visible, and no clinical adverse events were recorded. In the dissected vessels, the restenosis rate was significantly lower than in those without dissection (12% vs. 44%, p < 0.001); in the stented vessels, the restenosis rate was 25% (15/60). CONCLUSIONS In the presence of TIMI flow grade 3, coronary dissection is associated with a favorable outcome and predicts a low restenosis rate. These results caution against the indiscriminate use of intravascular prostheses in the event of nonocclusive coronary dissection.

Exercise-induced ischemic arrhythmias in patients with previous myocardial infarction: role of perfusion and tissue viability

OBJECTIVES This study sought to investigate whether residual viability of infarcted myocardium may play a role in the pathogenesis of exercise-induced ventricular arrhythmias. BACKGROUND We previously showed that transient ischemia within partially infarcted areas often precipitates ventricular arrhythmias during exercise that are consistently obliterated by intravenous nitrates. METHODS We studied 60 patients with chronic stable angina and a previous myocardial infarction. All underwent at least two consecutive exercise stress tests, coronary angiography and stress/rest myocardial perfusion tomography by Tc-99m 2-methoxy isobutyl isonitrile (MIBI). In the last 26 consecutive patients, residual viability was assessed by single-photon emission computed tomography (SPECT) using fluorine (F)-18 fluorodeoxyglucose. Perfusion and metabolic data were evaluated qualitatively by three independent observers in blinded manner. RESULTS With exercise, 30 patients (group A) consistently developed ventricular arrhythmias (> 10 ventricular ectopic beats/min, couplets, nonsustained ventricular tachycardia); the remaining 30 patients (group B) did not. The severity of coronary artery disease (Gensini score) was similar in the two groups. Postexercise SPECT showed partial reperfusion of an infarcted area in 28 of 30 patients of group A but in only 9 of 30 of group B (p < 0.0001). Uptake of F-18 fluorodeoxyglucose was observed within the infarcted zone in 10 of 13 and 1 of 13 patients in groups A and B, respectively (p = 0.0003). CONCLUSIONS In patients with myocardial infarction, exercise-induced ventricular arrhythmias appear to be triggered by transient ischemia occurring within a partially necrotic area containing large amounts of viable myocardium. Therefore, occurrence of arrhythmias during exercise may represent a clue to the presence of residual viability within a previously infarcted area.

Muramidase: A useful monocyte/macrophage immunocytochemical marker in swine, of special interest in experimental cardiovascular disease

The reliability of a rabbit polyclonal antibody against muramidase to identify monocytes/macrophages in swine was evaluated by immunostaining of cell smears and formaldehyde-fixed, paraffin-embedded tissue sections. Blood in tissue sections, cell smears (peripheral blood, buffy coat, and isolated mononuclear cells), and cultured mononuclear cells (adherent monocytes) contained positively stained cells with a morphology and in a number corresponding to that expected for a monocyte marker. Polymorphonuclear leukocytes (PMN), lymphocytes, and platelets were negative. In normal organs and tissues, mesenchymal cells with a distribution similar to that expected for macrophages were found to stain positively for muramidase. In pathologic tissues, positively stained inflammatory cells were identified in wounds, infected lungs, recently infarcted myocardium, and acute (variable numbers), organizing (often many), and healed (usually few) arterial thrombi. Enzymatic unmasking of antigenic determinants by trypsinization was necessary to achieve strong and consistent staining of monocytes/macrophages in tissue sections. A variety of epithelial cells of no differential diagnostic significance for monocyte/macrophage identification (e.g., renal proximal tubular cells) also stained positive for muramidase. The staining pattern of muramidase in swine corresponds to that described in humans, in whom muramidase has been shown to be a valuable marker of monocytes/macrophages. Swine PMN were, however, not stained or only weakly stained, whereas human PMN reportedly are strongly positive. As in humans, swine cardiac myocytes, smooth muscle cells, endothelial cells, lymphocytes, and platelets were consistently negative. This antibody against muramidase is a useful immunohistochemical marker for swine monocytes/macrophages in formaldehyde-fixed, paraffin-embedded tissues.

Protein S and protein C anticoagulant activity in acute and chronic cardiac ischemic syndromes. Relationship to inflammation, complement activation and in vivo thrombin activity

Protein S (PS) and protein C (PC) anticoagulant activities and thrombin-antithrombin complex (TAT) were measured in 20 patients with AIS, 25 patients with chronic stable angina (CSA) and a control group (C). Although plasma levels of TAT were significantly elevated in patients with CSA (p < 0.01 vs C), they were much higher in patients with AIS (p < 0.001 vs CSA). PC anticoagulant activity was similar in patients and controls. At variance, PS anticoagulant activity was lower in patients with AIS than in those with CSA and controls (p < 0.05), reflecting differences in total PS and C4B-binding protein (C4B-BP) antigen possibly resulting from involvement in the mechanisms of inflammation, complement activation and acute-phase response. The ratios of anticoagulant PS and PC to procoagulant vitamin K-dependent factors IX and II were reduced in AIS patients (0.05 > p > 0.005 vs C). In addition, the ratios of anticoagulant PC and PS to factor IX were lower in patients with AIS than in those with CSA (p < 0.05). These results indicate that in patients with acute ischemic cardiac syndromes the markedly increased in vivo thrombin generation is associated with an unbalance between coagulant and anticoagulant vitamin K-dependent factors.