Influence of Stent Length and Heparin Coating on Platelet Activation: A Flow Cytometric Analysis in a Pulsed Floating Model

doi:10.1016/S0049-3848(98)00198-4

Thrombosis Research

Volume 94, Issue 2, 15 April 1999, Pages 79-86

https://doi.org/10.1016/S0049-3848(98)00198-4 Get rights and content

Abstract

Platelets are involved in acute and subacute thrombotic occlusions of coronary stents and also may play a role in the pathophysiology of in-stent restenosis. This study sought to investigate the expression of activation dependent glycoproteins on platelets by flow cytometry and time until stent thrombosis in an in vitro model of stent thrombosis. Coronary stents were placed in parallel silicon tubings with circulating citrated platelet rich plasma to measure 1) influence of stent length on platelet antigens; 2) influence of heparin coating on platelet antigens; and 3) time until stent thrombosis. After recalcification aliquots of platelet-rich plasma were taken over 10 minutes in 2-minute intervals and immediately fixed and stabilized. For flow cytometric analysis monoclonal antibodies to CD41a (glycoprotein IIb/IIIa), CD42b (glycoprotein Ib-V-IX), CD62p (P-selectin), and CD63 (glycoprotein 53) were used. Within 2 minutes after start of circulation, the expression of CD62p and CD63 increased. Longer stents resulted in more platelet activation than shorter stents (25 mm vs. 15 mm; p<0.001. Time until stent thrombosis was reduced (25 mm vs. 15 mm; p<0.05). Heparin coating did not significantly influence flow cytometry detectable platelet activation but prolonged time until stent thrombosis (coated vs. uncoated; p<0.005). In control tubing systems without stents platelet activation was less pronounced (p<0.0001). Antibodies to CD41a and CD42b did not show significant changes. In this model platelet activation detected by flow cytometry and time until stent thrombosis were dependent on stent length and coating. In vitro testing could be useful to optimize stent design and material.

Section snippets

Materials and Methods

Blood samples were obtained from healthy and drug free volunteers (7 females, 5 males, mean age 28±4 years) via 16 G needles from antecubital veins into plastic syringes containing 3.8% sodium citrate solution (1:10). Platelet-rich plasma (PRP) was prepared and diluted to a final concentration of 250 platelets per nanoliter. To study changes of platelet antigens induced by stents (n=7 for each condition), a previously described in vitro model was used 9, 10. Stents were expanded with 12 atm for

Results

Structural antigens CD41a and CD42b did not show significant changes in all experiments. Within 2 minutes after recalcification and start of circulation the expression of activation dependent antigens CD62p and CD63 increased in all tubings with and without stents (control). Flow cytometric histograms of activated platelets showed an increasing right shift on the histogram x-axis, demonstrating higher values of forward-angle light scattering and FITC fluorescence than control platelet

Discussion

Endothelial and deep vessel injury during angioplasty induce platelet activation [16], thrombus formation, and activation of macrophages and smooth muscle cells. The resulting production and release of growth factors and cytokines may lead to a higher predisposition to restenosis [17]. The application of artificial devices into coronary arteries further contributes to an activation of platelets and implicates an additional thrombogenic potential [18]. Platelet activation by stents may be

Acknowledgements

We thank Jessika Preuß for her excellent technical assistance. We also thank Johnson & Johnson Interventional Systems, Norderstedt, Germany and Boston Scientific, Hilden, Germany who supported the study by providing the coronary stents.

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We have synthesized heparin-immobilized copolymers of l-lactide (LA) and 5-methyl-5-benzyloxycarbonate-1,3-dioxan-2-one (MBC) as non-inflammatory and non-thrombogenic biodegradable coating materials. These copolymers were used in fabricating arsenic trioxide (As₂O₃)-eluting stents to reduce the late-stage adverse events, such as thrombosis, localized hypersensitivity and inflammation, that occur when applying stents to treat coronary artery diseases. Heparinized copolymers effectively reduced platelet adhesion and protein adsorption while increasing the plasma recalcification time and thromboplastin time in vitro. Histological analysis of the polymer-coated stents in a porcine coronary artery injury model indicated that one heparinized copolymer (Hep-Co90, LA:MBC = 90:10), with the highest LA content of 90% and the lowest degradation rate, induced the least foreign body reactions and inflammation, which were as small as those induced by bare metal stents. Consequently, Hep-Co90 was used as the stent coating material for local As₂O₃ delivery. Histomorphometric evaluations suggested no significant difference between bare metal stents and As₂O₃-eluting stents at 1 and 3 months post-implantation. At 6 months, the lumen area in the porcine coronary arteries treated with As₂O₃-eluting stents is 32.4% higher than those treated with bare metal stents while the neointimal area, neointimal thickness and stenosis rate decreased by 25.8%, 32.5% and 31.2%, respectively. The As₂O₃-eluting stent using Hep-Co90 as the drug carrier and stent coating material presented in this study represents a novel promising device in preventing in-stent restenosis.
Restoration of Patency in Iliofemoral Deep Vein Thrombosis with Catheter-Directed Thrombolysis Does Not Always Prevent Post-Thrombotic Damage
2008, European Journal of Vascular and Endovascular Surgery
Citation Excerpt :
Among these, long stent placement was the only significant factor in a multivariate analysis and we consider that this should be avoided if possible. Although there have been no other reports about the effect of stent length on patency after CDT, it is reported in coronary interventions that a longer stent promotes platelet activation and increases re-stenosis.14,15 Sixteen of 34 patients developed post-thrombotic scarring of deep veins during the mean 42 month follow-up period.
To evaluate the long-term results of catheter-directed thrombolysis (CDT) and the feasibility of stent placement for lower extremity deep vein thrombosis (DVT).
Retrospective study of 34 patients (10 men and 24 women, mean age 55, S.D. 13 years) with lower extremity DVT underwent CDT at Seoul National University Hospital from January 1999 to October 2003. Patient characteristics, risk factors of DVT, extent of thrombosis, and short-term and long-term results of CDT and/or stent placement were analysed.
Mean follow-up times were 47 S.D. 16 months. The primary technical success rate was 97% (complete lysis 68%, partial 29%). During the follow-up periods 11 (32%) patients showed re-thrombosis. Sixteen (47%) of 34 patients showed chronic change of vessels during the follow-up periods. By Cox Proportional Hazard analysis, extent of thrombolysis was a statistically significant factor affecting the freedom of re-thrombosis and chronic change (P = 0.008 and P = 0.001). Nine (44%) of 21 deployed stents were obstructed, and the overall stent patency at 3 years was 56.7%. The only factor affecting the stent patency was stent length more than 6 cm (P = 0.002, HR 13, 95% CI 2.7–59).
Long-term results of CDT are not satisfactory because of the high recurrence rate of DVT and it cannot prevent chronic post-thrombotic damage to the affected vessels despite long-term anticoagulation therapy. Careful long-term surveillance of the venous function is highly recommended after CDT.
Flow patterns in an endovascular stent-graft for abdominal aortic aneurysm repair
2004, Journal of Biomechanics
Endovascular exclusion of the abdominal aortic aneurysm (AAA) has been carried out in selected patients during the past decade. The deployment of a complex multicomponent endovascular device in an aneurysmal aorta may alter the local haemodynamics and lead to thrombosis and intimal hyperplasia development. The aim of this in vitro study was to investigate the flow patterns using flow visualisation and laser Doppler anemometry in a commercial bifurcated stent-graft. Two configurations of the stent-graft, endo-stent and exo-stent, were investigated in an idealised planar AAA model. The flow structures in the main trunk in both configurations of the stent-graft are three-dimensional with complex secondary structures. However, these flow structures were not entirely caused by the stent-graft. The stent struts in the endo-stent configuration cause localised alteration in the flow pattern but the overall flow structures were not significantly affected. Low velocity regions in the main trunk and flow separation in the stump region and the curved segment of the iliac limbs were observed. These areas are associated with thrombosis in the clinical situation. Improvements in the design of endovascular devices may remove these areas of unfavourable flow patterns and lead to better clinical performance.
In vitro hemocompatibility studies of drug-loaded poly-(L-lactic acid) fibers
2003, Biomaterials
Our objective was to evaluate the hemocompatibility of biodegradable stent fibers, employing a closed-loop circulation system filled with human blood. We also investigated the effects of the anti-inflammatory and anti-proliferative drugs curcumin and paclitaxel, incorporated into stent fibers. Fresh whole blood was circulated in four parallel closed-loop systems: the empty tube circuit (control) and tubes containing either a PLLA fiber coil (PLLA), a curcumin-loaded PLLA coil (C-PLLA) or a paclitaxel-loaded PLLA coil (P-PLLA). The influence of PLLA fiber, alone or loaded with drug incorporated during melt-extrusion, on leukocyte and platelet adhesion and activation was determined by flow cytometry. The effects of blood flow and fiber properties on cell deposition were assessed by scanning electron microscopy (SEM). The flow cytometry results clearly demonstrated that PLLA triggers blood cell activation at the site of deployment, as shown by increases in CD11b, CD62P and leukocyte–platelet aggregates, compared to controls. Curcumin and paclitaxel treatments both significantly reduced leukocyte and platelet activation and adhesion to PLLA fibers, as shown by flow cytometry and SEM. Activated leukocytes and platelets revealed significantly lower CD11b and CD62P receptor binding for C-PLLA compared with PLLA alone, and slightly lower for P-PLLA. Reductions in platelet–leukocyte aggregates were observed as well. In addition, there was less leukocyte and platelet adhesion to C-PLLA, compared with PLLA fiber controls, as shown by SEM. A continuous linear thrombus, composed of platelets, leukocytes, red blood cells and fibrin was occasionally detected along the line of tangency between the coil and the tube wall. Flow separation and eddying, proximal and distal to the line of tangency of coil and tube, is thought to contribute to this deposit. Curcumin was more effective than paclitaxel in reducing leukocyte and platelet activation and adhesion to PLLA stent fibers in this setting. However there was evidence of paclitaxel degeneration during melt extrusion that may have inhibited its effectiveness. Incorporation of the anti-inflammatory and anti-proliferative drug curcumin into bioresorbable stent fibers is proposed to prevent thrombosis and in-stent restenosis.
Fabrication of drug-eluting covered stents with micropores and differential coating of heparin and FK506
2003, Cardiovascular Radiation Medicine
To reduce in-stent restenosis rates, we developed a novel drug-eluting covered stent with a microporous elastometric covered film, in which its luminal surface was flat and immobilized with heparin for anticoagulation and its outer surface immobilized with FK506 to prevent neointimal hyperplasia. One month after implantation into the bilateral common carotid arteries, all stented arteries were patent and the luminal surfaces were fully covered with a confluent of endothelial cells irrespective of the drug immobilization. In the control group, which consisted of covered stents without drug immobilization, intensive inflammatory cells adjacent to the stents and neointimal hyperplasia, indicating vascular injury, were observed. In contrast, in the developed drug-eluting stents, only a few inflammatory cells around the stent strut and covered film were observed, and there was no significant neointimal thickening.
Heparin-coated balloon-expandable stent reduces intimal hyperplasia in the iliac artery in baboons
2003, Journal of Vascular and Interventional Radiology
To examine the effect of heparin-coated balloon-expandable iliac stent placement on intimal hyperplasia in a baboon model.
Balloon-expandable (Palmaz-Schatz) stents were placed in bilateral common iliac arteries in 20 male baboons (mean weight: 8.8 kg). In each animal, a heparin-coated iliac stent was placed on one side and the contralateral iliac artery received an uncoated stent that served as a control. The iliac artery stents were harvested at 30 days (n = 10) and 90 days (n = 10). Arteriography was performed to assess iliac patency and intravascular ultrasonography was used to determine neointimal and luminal areas. Histologic and morphometric analysis and scanning electron microscopy were performed in the stent-implanted iliac arteries.
One animal was excluded in the 30-day group because of premature death. In the remaining nine surviving animals, seven control stents (78%) and nine heparin-coated stents (100%) remained patent. Morphometric analysis showed that the iliac arteries with heparin-coated stents had larger luminal areas (17%, P < .05), less neointimal area (26%, P < .05), lower neointima-to-media ratios (32%, P < .05), and equivalent medial areas (P = .92) compared to the control group at 30 days. In contrast, all control and heparin-coated stents were patent (100%) in the 90-day group. In that group, the heparin-coated stent group had less neointimal area (28%, P < .05), lower neointima-to-media ratios (42%, P < .05), and equivalent medial area (P = .92) and luminal area (P = .07) compared to the control group.
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Regular articleInfluence of Stent Length and Heparin Coating on Platelet Activation: A Flow Cytometric Analysis in a Pulsed Floating Model

Abstract

Section snippets

Materials and Methods

Results

Discussion

Acknowledgements

J Am Coll Cardiol

Thromb Res

Throm Res

Am J Cardiol

J Biol Chem

J Am Coll Cardiol

Lancet

Intravascular stents to prevent occlusion and restenosis after transluminal angioplasty

N Engl J Med

A comparison of balloon-expandable stent implantation with balloon angioplasty in patients with coronary artery disease

N Engl J Med

A randomized comparison of coronary stent placement and balloon angioplasty in the treatment of coronary artery disease

N Engl J Med

Subacute stent thrombosisEvolving issues and current concepts

J Am Coll Cardiol

A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronary artery stents

N Engl J Med

Platelet membrane activation markers are predictive for increased risk of acute ischemic events after PTCA

Circulation

Platelet activation and coronary stent implantation

Circulation

Heparin-coated Palmaz-Schatz Stents in human coronary arteries. Early outcome of the BENESTENT-II pilot study

Circulation

Reduction in thrombotic events with heparin-coated Palmaz-Schatz stents in normal porcine coronary arteries

Circulation

Multiple stents increase target vessel revascularization rates

J Am Coll Cardiol

Multivessel stenting without anticoagulationImmediate and short-term outcome

Eur Heart J

Regular article
Influence of Stent Length and Heparin Coating on Platelet Activation: A Flow Cytometric Analysis in a Pulsed Floating Model