Collagen-Induced Thrombus Formation in Flowing Nonanticoagulated Human Blood From Habitual Smokers and Nonsmoking Patients With Severe Peripheral Atherosclerotic Disease
Abstract The objective of the present study was to investigate collagen-induced platelet thrombus formation at arterial blood flow conditions in nonanticoagulated blood from habitual smokers and from nonsmoking patients with severe peripheral atherosclerotic disease. Collagen-induced thrombogenesis was elicited in native blood drawn directly from an antecubital vein over immobilized type III collagen fibrils coated on a coverslip positioned in a parallel-plate perfusion chamber. The wall shear rates at the collagen surface were comparable to those encountered in medium-sized (650 s−1) and moderately stenosed (2600 s−1) arteries. Thrombus formation in blood from habitual smokers after 10 hours of smoking abstinence appeared to be not different from thrombus formation in blood from healthy nonsmokers. However, immediately after a cigarette had been smoked, thrombus volume in blood from the same individuals was increased twofold at the highest shear rate (P<.05). Thus, the thrombotic response was temporarily upregulated after smoking. Thrombus formation in blood from nonsmoking patients with severe peripheral atherosclerotic disease was neither enhanced nor decreased but was within the range of the nonsmoking control subjects. However, fibrinopeptide A generation after 4 minutes of perfusion at 2600 s−1 was higher in blood from the atherosclerotic patients (P<.05) and associated with a higher plasma fibrinogen level (P<.005). Thus, signs of changed platelet reactivity in flowing nonanticoagulated blood were encountered only in the habitual smokers immediately after they had smoked a cigarette.
- cigarette smoking
- intermittent claudication
- peripheral arterial disease
- arterial blood flow
- thrombus formation
- Received July 18, 1994.
- Accepted October 31, 1994.
Thrombotic disorders are closely associated with atherosclerosis.1 It is generally accepted that platelets play a pivotal role in both processes,2 particularly in acute thrombosis. Abnormalities of platelet function have been associated with various “prethrombotic” disorders, although attempts to demonstrate hyperreactive platelets in patients have yielded sparse information.3 Most of the studies revealed only minor or inconsistent abnormalities. The goal of the present study was to investigate and characterize thrombus formation at arterial blood flow conditions in nonanticoagulated human blood from two groups with “prethrombotic risk factors,” ie, habitual cigarette smokers and patients with severe peripheral atherosclerotic disease. In both groups, biochemical evidence exists for in vivo platelet activation with enhanced thromboxane A2 production.4 5
Platelet reactivity is most often characterized through the response of platelets to an external agonist. To study platelet reactivity in flowing nonanticoagulated blood from these individuals, we used a parallel-plate perfusion device.6 7 Thrombogenesis was elicited by immobilized type III collagen fibrils, and platelet-collagen adherence and platelet thrombus formation on the collagen surface were quantified. This model was previously validated by correlation with simultaneously induced experimental in vivo coronary thrombosis in dogs.8 Other results obtained with this model correlate well with clinical findings in humans, eg, in patients with factor VIII deficiency and various subtypes of von Willebrand’s disease.9 Of particular interest to the present study is the fact that “acute” cigarette smoking increases the thrombotic response at high arterial shear conditions10 and that platelet-collagen adhesion is enhanced in blood from patients with hyperlipoproteinemia IIa and IIb under similar blood flow conditions.11
Twenty male volunteers were included in this open study. None of the volunteers had taken aspirin or other nonsteroidal anti-inflammatory drugs for at least 10 days before the blood donations. All individuals were fully informed about the study and had given free, informed consent for participation. The protocol was approved by the local ethics committee and the study conducted according to the principles of the Declaration of Helsinki.
Nonsmoking Control Subjects
Seven healthy nonsmokers were recruited.
Six patients with angiographically documented severe peripheral atherosclerotic arterial disease referred to the vascular surgery unit of Ullevaal University Hospital were entered into the study. All suffered from severe intermittent claudication, with pain at rest or walking a distance less than 50 m. Two of the patients had a previous history of myocardial infarction. The patients claimed not to have smoked during the previous 2 years, and they were not suffering from diabetes mellitus or other chronic diseases.
Two of the patients were taking pentoxifylline up to 24 hours before the perfusion experiment. The elimination half-life of pentoxifylline given as a conventional sustained-release tablet formulation is 3.4 hours.12 Since there were no differences between these two patients and the rest of the group, their data were retained in the analysis.
Seven healthy cigarette smokers were recruited. They were all habitual smokers, smoking at least 10 cigarettes a day for more than 5 years.
Abstaining smokers. Abstaining smokers were willing to abstain from cigarette smoking during a 10-hour period before the blood donations.
Acute smoking. In a separate set of experiments, the same habitual cigarette smokers smoked one cigarette within 5 minutes before blood donations for perfusion experiments at a wall shear rate of 2600 s−1.
The acute effects of cigarette smoking in the ex vivo thrombosis model were recently reported in detail.10 From these studies it appears that smoking increases the thrombotic response only at 2600 s−1, and therefore we considered it unnecessary to repeat the studies on acute effects at 650 s−1.
After venipuncture with a No. 19 Butterfly Infusion Set (Abbott Laboratories), the first 5 mL of blood was collected into EDTA for determination of platelet count and hematocrit (Auto Counter AC 920, Swelab Instruments). The following 2.9 mL of blood was collected into two Eppendorf tubes, one prefilled with 0.15 mL of 129 mmol/L sodium citrate for determination of plasma fibrinogen and serum thiocyanate.
Plasma Fibrinogen and Serum Thiocyanate
Plasma fibrinogen levels were quantified in citrated plasma according to Clauss.13 Clotting time was recorded with a coagulometer KC10A (Amelung GmbH), and a standard curve was prepared with human fibrinogen (Baxter Dade AG).
Thiocyanate was measured spectrophotometrically directly in serum as described by Degiampietro and Peheim,14 the method being adapted to a Cobas-Bio centrifugal analyzer (Hoffmann–La Roche Ltd).
Ex Vivo Perfusion Experiments
Ex vivo perfusion experiments15 were performed at 37°C with collagen-coated coverslips positioned in parallel-plate perfusion chambers.6 16 Purification and fibrillar formation of collagen type III were previously reported in detail.17 18 19 The fibrillar collagen suspension was spray-coated19 onto washed Thermanox plastic coverslips (Miles Laboratories) to a final density of approximately 20 μg/cm2. A density of 10 μg/cm2 gives a maximal thrombogenic stimulus.20
After venipuncture and collection of blood samples, the next 50 mL of nonanticoagulated blood was drawn directly over the collagen surface by an occlusive roller pump (Gilson model M312) at a constant flow rate of 10 mL/min. The pump was placed distal to the perfusion chamber. Wall shear rates characteristic of healthy medium-sized (650 s−1) and moderately stenosed arteries (2600 s−1) were maintained at the collagen surface for 5 minutes. The different cross-sectional dimensions of the rectangular blood flow channel and the blood flow rate determine the wall shear rate.
Platelet-collagen adherence, fibrin deposition, and thrombus volume on the collagen surface were quantified by light microscopy.21 Evaluations were performed on semithin sections (1 μm) prepared perpendicular to the direction of the blood flow 1 mm downstream from the upstream edge of the coverslip.22 The sections were stained with basic fuchsin and toluidine blue.21
Standard morphometry was used to assess the percentage of the surface covered with platelets (percent platelet adhesion) and with fibrin (percent fibrin deposition).21 The evaluations were performed with a Zeiss Standard 25 light microscope at ×1000 magnification.
Thrombus area (micrometers squared per micrometers of sectional length) was assessed by computer-assisted morphometry (Kontron Vidas image-analysis unit, Zeiss). Thrombus volume (micrometers cubed per micrometers squared) was derived from the sectional thrombus area as previously described.22 The evaluations were carried out at ×500 or ×2000 magnification, depending on thrombus size.
Fibrinopeptide A and β-Thromboglobulin
Plasma levels of fibrinopeptide A (FPA) and β-thromboglobulin (β-TG) were measured distal to the perfusion chamber.7 At 4 minutes of perfusion time, blood samples (2×0.9 mL) were collected immediately distal to the chamber, as previously described in detail.23 The samples were collected successively during 15 seconds into syringes prefilled with 0.1 mL of 1000 IU heparin plus 1000 kallikrein inhibiting units Trasylol per milliliter of saline for FPA and an anticoagulant mixture according to Ludlam and Cash24 for β-TG. The samples were immediately chilled on ice. Further processing was done according to the manufacturers of the respective kits (FPA from IMCO and β-TG from Amersham).
The significance of differences between groups was calculated with the Mann-Whitney U test to avoid any assumptions about the distribution of the material. Values of P<.05 were considered significant.
Hematocrit, Hemoglobin, and Platelet Count
The group of habitual cigarette smokers had higher platelet counts than the nonsmoking control subjects (P<.05). Hematocrit and hemoglobin were significantly lower in the atherosclerotic patients (Table 1⇓).
Plasma Fibrinogen and Serum Thiocyanate Levels
A higher plasma fibrinogen level was observed in the atherosclerotic patients relative to the plasma levels of nonsmokers (P<.005) and habitual smokers (P<.05) (Table 1⇑).
Serum thiocyanate levels were less than 70 μmol/L in all nonsmokers and atherosclerotic patients, indicating that the information about their smoking habits was correct. The mean serum thiocyanate level of the habitual smokers exceeded 70 μmol/L and was significantly higher than the serum levels in nonsmokers (P<.05) and atherosclerotic patients (P<.005) (Table 1⇑).
The average thrombus volume at the wall shear rate of 650 s−1 was slightly lower in blood from the atherosclerotic patients although not significantly different from the volumes observed in blood from nonsmoking control subjects and habitual smokers (Table 2⇑).
The thrombus volume at 2600 s−1 was nearly twofold increased in blood from cigarette smokers immediately after smoking one cigarette, being significantly different from both atherosclerotic patients (P<.01) and habitual smokers after 10 hours of smoking abstinence (abstaining smokers) (P<.05) (Table 3⇑). However, no significant differences were found between nonsmokers, atherosclerotic patients, and abstaining smokers. Thus, the thrombus volume in blood from habitual smokers was temporarily increased after the smoking of a cigarette.
β-TG Plasma Levels
The median β-TG plasma levels were not different between the various groups at 650 s−1 (Table 4⇓). In contrast, at 2600 s−1 a significant difference in the median β-TG plasma levels (P<.05) was observed between nonsmokers and abstaining smokers (Table 5⇓). However, the increased thrombus formation immediately after cigarette smoking was not reflected in an increased platelet release of β-TG.
Fibrin Deposition on Collagen and FPA Plasma Levels
The median FPA plasma levels did not vary between the different groups at 650 s−1 (Table 4⇑). However, a significant difference (P<.05) in median FPA plasma levels was observed between nonsmokers and atherosclerotic patients at 2600 s−1 (Table 5⇑).
Platelets play an important role in the development of atherosclerosis and its thromboembolic complications.2 25 In thromboembolic disorders, the complex interplay between the vessel wall, platelets, and plasma proteins is strongly affected by local blood flow conditions.26 The goal of the present study was to investigate collagen-induced arterial thrombus formation in blood from groups of volunteers at risk to develop thrombotic disorders, such as habitual cigarette smokers and patients with severe peripheral atherosclerotic disease. The study was performed with a human ex vivo model of thrombogenesis6 7 at wall shear rates comparable to those encountered in medium-sized (650 s−1) and moderately stenosed (2600 s−1) arteries.
Thrombus formation in blood from habitual cigarette smokers was not enhanced after 10 hours of smoking abstinence. However, the smoking of a cigarette increased thrombus volume twofold at the highest arterial shear condition (2600 s−1). Thus, the thrombotic response was temporarily and reversibly upregulated by “acute” cigarette smoking. Previous findings have shown that this increased thrombus formation is due to enhanced platelet reactivity mediated through thromboxane A2, since aspirin reduces the thrombotic response to the level observed in healthy nonsmoking individuals.10
Evidence for changed platelet reactivity in patients with severe peripheral atherosclerosis was not found, since both platelet-collagen adhesion and thrombus volume were not different from the levels observed in nonsmoking control subjects. Also, the postchamber β-TG plasma levels indicated a normal platelet reactivity at both shear rates. To eliminate the possible confounding effects of smoking, all the atherosclerotic patients participating in this study were currently nonsmokers. However, it should be emphasized that cigarette smoking is considered to be one of the major risk factors for developing intermittent claudication.27
Several prospective epidemiological studies have demonstrated a positive statistical correlation of plasma fibrinogen concentration with subsequent cardiovascular events after adjusting for other known risk factors.28 29 In our study the atherosclerotic patients had an elevated plasma fibrinogen level. At both shear rates, FPA generation after 4 minutes of perfusion was higher in the atherosclerotic patients, reaching significance versus nonsmokers at 2600 s−1. However, there was no individual correlation between the plasma fibrinogen level and platelet thrombus formation or the low fibrin deposition on the collagen surface.
Platelet aggregation is in general the precipitating event in acute arterial thrombosis. An increased risk for thrombosis in nonsmoking atherosclerotic patients apparently cannot be explained by an altered platelet function. However, persistent alterations in the endothelial lining of the vessels with defective defense mechanisms and/or disturbed blood flow patterns introduced by stenotic lesions may promote thrombogenesis.26 Atherosclerotic patients in general are prone to experience plaque ruptures,30 which may result in exposure of thrombogenic components such as tissue factor and collagen to the bloodstream.
The present results show that arterial ex vivo thrombus formation in blood from cigarette smokers is reversibly upregulated immediately after smoking and that the platelet thrombus formation is within the “normal” range after 10 hours of smoking abstinence. Patients with severe peripheral atherosclerotic disease who do not smoke show a collagen-induced platelet thrombus formation at arterial blood flow conditions with no signs of platelet hyporeactivity or hyperreactivity.
The study was in part financially supported by Johan and Ole Ekeberg’s Medical Research Fund, Alexander Malthe’s Legacy, Nelsons Fund, and Professor Carl Semb’s Medical Research Fund. This work was performed at Ullevaal University Hospital and Nycomed Bioreg AS. We want to acknowledge the staff at the Peripheral Vascular Unit, Department of Surgery, Ullevaal University Hospital, for their kind support, and Dr Peter Urdal, Ullevaal University Hospital, for help with the thiocyanate analysis. Dr Carl-Erik Slagsvold, Aker University Hospital, is acknowledged for help in recruiting patients with intermittent claudication.
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