In vitro model to test the thrombogenicity of coronary stents.

Thrombotic occlusion is a major complication limiting the application of stents in coronary arteries. In an in vitro model we investigated the thrombogenicity of different stent materials and several medical regimens to prevent thrombotic occlusion. Experiments were conducted in a closed system of silicon tubing with circulating citrated platelet rich plasma of healthy volunteers (n = 7) and of patients (n = 7 for each condition). Patients were either treated with phenprocoumon or with high or low dose heparin in combination with aspirin alone (100 mg) or aspirin (990 mg) plus dipyridamole (225 mg). After placement of tantalum wire stents into the system platelet aggregates were visible after 13.5 +/- 3.0 min, and occlusion occurred after 15.0 +/- 3.5 min. Similarly, with implanted stainless steel stents aggregation was seen after 13.0 +/- 3.5 min and thrombosis occurred after 14.5 +/- 3.5 min (p < 0.001 vs control without stent). Microscopic examination revealed combined platelet fibrin thrombi occluding the lumen. Platelet components predominately covered stent wires, particularly at crossing points. In all experiments high-dose heparin prevented platelet aggregate formation and stent occlusion independently of additional aspirin or aspirin plus dipyridamole; perfusion time > 60 min (p < 0.001 vs no heparin). Low-dose heparin could not prevent clotting. With aspirin alone aggregates were visible after 16.0 +/- 4.0 min and clotting occurred after 23.0 +/- 5.0 min. In combination with dipyridamole aggregates were visible after 15.5 +/- 5.0 min and clotting after 21.0 +/- 4.0 min (NS vs aspirin alone). Phenprocoumon prevented platelet aggregate formation and stent occlusion; perfusion time > 60 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of coronary stent-induced alterations of platelet antigens in an in vitro model.

One of the limitations of coronary stenting is the subacute thrombotic occlusion. In an in vitro model, we examined the effects of tantalum wire stents (n = 12) on platelet antigens. Platelet-rich plasma (PRP) was circulated in PVC tubing systems. At fixed intervals over a 10-min time course, aliquots of PRP were drawn, stained with monoclonal antibodies (CD41a, CD42b, CD62p, and CD63), and analyzed by flow cytometry. Within 2 minutes of the onset of circulation, expression of the activation-dependent antigens CD62p and CD63 increased in all tubing systems with stents. This early increase was followed by a progressive rise in fluorescence intensity of these neoantigens over the course of 10 minutes (p < 0.05 vs.. control system without stent). Antigens CD41a and CD42b did not show significant changes in either system. The artificial surfaces and shear forces of stent meshes induce alterations in platelet antigens. Flow cytometry provides a sensitive technique for in vitro testing of the thrombogenicity of coronary stents, and may be useful in further improving stent biocompatibility.

In vitro analyses of diamond-like carbon coated stents. Reduction of metal ion release, platelet activation, and thrombogenicity.

Coronary artery stents can induce platelet activation by shear forces, contact to the biomaterial, and release of metal ions. This activation is one important trigger for thrombosis. Coating of stents is a possible approach to prevent this side effect. The purpose of this study was to evaluate in vitro the biocompatibility of stents coated with diamond-like carbon (DLC). Semiquantitative energy-dispersive X-ray microanalyses showed a complete coverage of the DLC stents. Flow cytometric analyses revealed a significantly higher increase of mean channel fluorescence intensity for the activation-dependent antigens CD62p and CD63 in non-coated compared to DLC-coated stents (p<0.05). Atomic adsorption spectrophotometry analyses revealed a significant release of nickel and chromium metal ions by non-coated stents over a storage period of 96 hours in human plasma (p<0.05). In contrast, only minimal concentrations of released ions could be detected in the case of DLC-coated stents. Similar observations were made with inductively coupled plasma mass spectrometry analyses. Here, high concentrations of molybdenum and manganese ions were released from non-coated stents (p<0.05), while release of these ions from DLC-coated stents was virtually undetectable (p=0.1 for molybdenum and p=0.4 for manganese). Coating of intracoronary stents with diamond-like carbon significantly improves biocompatibility. This biocompatible coating may therefore contribute to a reduction in thrombogenicity.

Influence of stent length and heparin coating on platelet activation: a flow cytometric analysis in a pulsed floating model.

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.

Platelet hemostasis capacity in smokers. In vitro function analyses with 3.2% citrated whole blood.

INTRODUCTION: Platelet function may be influenced by cigarette smoking. We therefore examined the effect of smoking on platelet hemostasis capacity (PHC) with an in vitro analyzer (PFA-100). METHODS AND RESULTS: Healthy blood donors (n=54) were included in the study and divided into four groups: nonsmoking males (n=14), nonsmoking females (n=14), smoking males (n=12) and smoking females (n=14). For in vitro analyses, in each participant citrated blood (3.2% buffered) was tested for PHC by two cartridges coated with collagen, and additionally with epinephrine (Col/Epi) or ADP (Col/ADP). Analyses were performed within 4 h after sample taking. PHC was expressed as the time in seconds to occlude the cartridge (closure time, CT). The average CT was significantly prolonged in female smokers compared to the female nonsmoking group for both types of cartridges (Col/Epi: P=.02; Col/ADP: P=.03). No significant differences were detected comparing the CT of smoking and nonsmoking males. After pooling male and female smokers and nonsmokers, no significant differences could be found, neither for the Col/Epi cartridges nor the Col/ADP cartridges. Plaletet aggregation assays performed in parallel showed no significant differences, except a reduced aggregability in male smokers compared to male nonsmokers using epinephrine 8.0 microM/ml as activating agent (P=.01). Furthermore, smoking volunteers presented with a significantly increased fibrinogen level compared to nonsmoking volunteers (P<.01). CONCLUSIONS: The results of our study show that in habitual smokers PHC (PFA-100) and the capability of platelets to react upon agonist stimulation in aggregation assays is not significantly influenced or increased compared to healthy nonsmokers. However, an immediate effect of cigarette smoking cannot be excluded.

[Elective percutaneous tracheotomy in long-term ventilation in a coronary intensive care unit]. / Elektive perkutane Tracheotomie bei langzeitbeatmeten Patienten auf einer kardiologischen Intensivstation.

BACKGROUND AND OBJECTIVE: Long-term mechanical ventilation presents a problem in an intensive care unit (ICU). If done via oral intubation it brings the danger of bacterial contamination and the development of pressure lesions of the gums and tongue. Furthermore, the tube is usually tolerated for only a short time during the weaning phase. Nasal intubation is associated with an increased incidence of nasal necroses and sinusitis. Tracheal intubation has the advantage that the dead space is reduced, it is more comfortable for the patient and the weaning period is shortened, thus decreasing the duration of bed confinement. Percutaneous tracheostomy (PT) was introduced as an alternative to conventional surgical tracheostomy. It was the aim of this study to present the authors' experience with PT. PATIENTS AND METHODS: PT was performed under bronchoscopic control with the aid of dilators or dilatating forceps in 78 patients in a cardiological ICU (13 women, 68 men; average age 64 +/- 14 years) with heart failure (n = 34 [44%]), cerebral problems post-resuscitation (n = 32)[41%]), pulmonary infection (9[11%]) or other conditions (n = 3[4%]). RESULTS: Because of contraindications PT was not done in 12 patients (15%). Percutaneous emphysema developed in one woman as a result of injury to tracheal cartilage. There were no other complications. Two patients with thrombocytopenia were given platelet concentrates during the PT. At the end of the period of ventilation the tracheostomy tube was removed. Spontaneous closure of the stoma occurred within 3 to 5 days. CONCLUSIONS: PT is a minimally invasive alternative to surgical tracheostomy. It has few complications and can be performed in a cardiological ICU.

Effects of aspirin and prostaglandin E1 on in vitro thrombolysis with urokinase. Evidence for a possible role of inhibiting platelet activity in thrombolysis.

The formation of thrombi in vivo includes the activation of both platelets and the coagulation cascade. Conventional thrombolytic therapy is primarily directed toward the dissolution of fibrin. To evaluate the possibility that platelet activity impairs the lysis of thrombi, we studied the effects of aspirin and platelet-deaggregating prostaglandin E1 on thrombolysis with urokinase. Combined platelet and fibrin thrombi were produced in vitro by adding CaCl2 and collagen (1 microgram/ml) to citrated platelet-rich plasma (250,000 platelets per microliters). Urokinase (500-10,000 units/ml) caused a dose-dependent weight loss of the thrombi that was maximal at 2,000 units/ml. The addition of aspirin (10-200 micrograms/ml) to platelet-rich plasma before thrombus formation markedly enhanced thrombolysis with urokinase. This effect was most pronounced at 20 micrograms/ml aspirin. However, when aspirin was added after completion of thrombus formation, no significant effect on thrombolysis was noted. Prostaglandin E1 (1-100 mumol/l) improved the lysis with urokinase of the combined platelet and fibrin thrombi. This effect was maximal at 20 mumol/l prostaglandin E1. When pure fibrin thrombi were produced in platelet-free plasma, prostaglandin E1 was without effect on lysis. Thus, in vitro lysis with urokinase of combined platelet and fibrin thrombi was enhanced by the addition of platelet-deaggregating prostaglandin E1 and by pretreatment with aspirin.

[Thrombus age as a determinant of lysis efficacy of in vitro produced platelet-fibrin thrombi]. / Das Thrombusalter als Determinante der Lysierbarkeit von in vitro hergestellten Plättchen-Fibrin-Thromben.

Thrombus age as a determinant of lysis efficacy has rarely been investigated. We developed an in vitro model to study the lysis of collagen induced platelet-fibrin-thrombi of different ages. Histologically, the thrombi were similar to those found in coronary vessels of patients deceased from acute myocardial infarction or unstable angina. After 10 (TA10), 30(TA30) und 60 (TA60) min of thrombus aging thrombolysis over 30 min with urokinase alone or in combination with prostaglandin E1 was started. The efficacy of lysis with urokinase alone decreased with increasing thrombus age. After 30 min of lysis with urokinase alone weight of TA10-thrombi was reduced by 16.4 +/- 1.2 mg, of TA30-thrombi by 11.5 +/- 0.9 mg (p < 0.001 vs TA10-thrombi) and of TA60-thrombi by 7.8 +/- 1.1 mg (p < 0.001 vs TA30, p < 0.0001 vs TA10-thrombi). The addition of prostaglandin Ei augmented lysis of TA10-thrombi, after 30 min of lysis thrombus weight was reduced by 17.3 +/- 1.2 mg (p < 0.01 vs urokinase alone). Lysis of 30 and 60 min old thrombi was nearly unaffected. Thus, thrombus age is a strong predictor of lysis efficacy of in vitro collagen induced platelet-fibrin-thrombi.

Tantalum stents induce expression of GMP140 and GP53 on platelets after contact in an in vitro model.

Endovascular implantation of stents has become increasingly important to reduce acute complications and restenosis after percutaneous transluminal coronary angioplasty (PTCA). Nevertheless, the risk of stent related thrombosis still appears to be a limitation in this therapeutic approach. As platelets play a major role in thrombus formation, we examined platelets in an in vitro model by flow cytometry to assess the expression of activation-dependent epitopes caused by shear stress and contact to artificial surfaces of stents. Tantalum wire stents (n = 12) were placed in one of two parallel silicon tubing systems. Both systems were filled with citrated platelet-rich plasma (PRP) of healthy and drug-free volunteers. After recalcification, aliquots of PRP were drawn via a three-way faucet over a course of 10 min. For flow cytometric analysis monoclonal antibodies CD41a, CD42b, CD62p and CD63 were applied. Within 2 min after onset, the expression of CD62p (GMP140) and CD63 (GP53) increased in the tubing system with the stent. Over the course of 10 min, platelet activation progressively increased (CD62p p < 0.05 vs. control system without stent; CD63 p < 0.005 vs. control). Antigens CD41 a and CD42b did not show significant changes in both systems. Artificial surfaces and shear forces of stent meshes may contribute to the activation of platelets, thereby promoting the thrombotic potential. As an utmost sensitive diagnostic tool, flow cytometry may attribute to further improvement of material and design of stents.

[Flow-cytometric analysis of platelet activation during rotablation]. / Durchflusszytometrisch nachgewiesene Aktivierung von Thrombozyten im Verlauf der Rotablation.

Acute occlusion and subacute restenosis of the coronary artery are still the limiting factors of the otherwise successful interventional cardiology. Platelets and especially activated platelet populations play a key role concerning these typical and sometimes fatal complications. In this study we used flow-cytometry to determine the influence of the modern interventional technique of rotablation on platelet antigens and their possible alteration. A PTCA control group was included. We analyzed the fluorescence expression of structural antigens CD41a (GPII-IIIa) and CD42b (GPIb-V-IX), and of the activation-dependent antigens CD62p (P-selectin, PADGEM, GMP-140) and CD63 (GP53). Furthermore we analyzed the binding of fibrinogen to the platelet flow-cytometrically. CD41a and CD42b did not show significant alternations in fluorescence before, directly after and thirty minutes after finishing PTCA and rotablation (PTCA: CD41a p=0.8 and 0.9; CD42b p=0.5 and 0.2; rotablation: CD41a p=0.2 and 0.2; CD42b p=0.4 and 0.1). But platelet activation could be detected directly after PTCA and rotablation measuring the mean channel fluorescence intensity (MCFI) of CD62p, CD63 and fibrinogen binding (all p<0.05). Thirty minutes after finishing the procedures there were again significant changes in MCFI in PTCA (CD62p, CD63, fibrinogen binding; all p<0.05), but not in rotablation (CD62p p=0.1; CD63 p=0. 9; fibrinogen binding p=0.5). But MCFI for CD62p and fibrinogen binding in rotablation was higher than in PTCA. The results of our study show that rotablation also induces significant platelet activation that is higher than in PTCA alone. Flow cytometry is a sensitive and specific, multiparametric tool in establishing platelet activation. The individual platelet activation process is part of a complex cascade of events happening in the rotablated coronary segment leading to a vascular-molecular inflammatory process and consecutive clinical problems in some patients.

Multicenter evaluation of the Strecker tantalum stent for acute coronary occlusion after angioplasty.

The Strecker stent is a balloon-expandable, flexible endoprosthesis constructed of knitted tantalum wire and has been implanted successfully in peripheral arteries. This study presents the first multicenter experience with implantation of this radiopaque device in the coronary arteries in 64 patients of 6591 consecutive percutaneous transluminal coronary balloon angioplasty (PTCA) procedures complicated by abrupt closure. In all except 1 patient the stents (n = 72) were correctly placed, and flow could be reestablished immediately. During hospitalization 12 (19%) patients had stent closures; 5 (8%) patients had Q-wave myocardial infarctions; and 13 (20%) patients underwent bypass surgery (4 on an emergency basis). The in-hospital mortality was 9%: 2 patients died after thrombotic stent occlusions; 2 patients had fatal bleeding complications; and 2 patients died after bypass surgery. Major bleeding complications at the puncture site were observed in 8 (12.5%) patients. Angiograms (n = 45) after 17 +/- 5 weeks revealed a stent patency rate of 89%. Thus the Strecker coronary stent proved to be helpful in the management of acute vessel closure during PTCA. However, in this first series a high incidence of early thrombotic occlusions and bleeding complications warrants close anticoagulation monitoring and limits broader indications.

[Peripheral embolism of hemostasis collagen (VasoSeal)]. / Periphere Embolie von Hämostasekollagen (VasoSeal).

VasoSeal is a purified bovine, absorbable collagen plug currently successfully used to close the femoral arterial puncture site after cardiac catheterization under full anticoagulation. Up to now there has been no experience with potential complications. We observed acute ischemia in the right lower leg of 2/100 patients 36 resp. 24 h after successful closure of the puncture site with VasoSeal. Angiography confirmed acute occlusion of the distal A. poplitea dextra. A 25-mm resp. 50-mm long cylindrical foreign body embolus was removed with a Fogarty-catheter by retrograde indirect embolectomy. Histopathology confirmed a fresh collagen clot with appositional thrombosis.