- Email: [email protected]
Platelet Reactivity in Patients With a History of Obstructive Prosthetic Valve Thrombosis
Platelet Reactivity in Patients With a History of Obstructive Prosthetic Valve Thrombosis Tal Bouganim, MD, Yaron Shapira, MD, Alexander Sagie, MD, Mordehay Vaturi, MD, Alexander Battler, MD, Ran Kornowski, MD, and Eli I. Lev, MD* One of the most serious complications of mechanical valves is obstructive prosthetic valve thrombosis (OPVT or “stuck valve”). Some patients develop OPVT despite an international normalized ratio (INR) in the therapeutic recommended range. We hypothesized that patients who develop OPVT have hyper-reactive platelets. We, therefore, examined platelet reactivity in patients who developed OPVT, despite a therapeutic or near-therapeutic INR, compared with a matched control group. We compared platelet reactivity between patients who had had an OPVT episode, despite a therapeutic or near-therapeutic INR (n ⴝ 18), and a matched group of patients with mechanical valves who did not develop this complication (n ⴝ 18) from 1996 to 2007. Platelet reactivity was evaluated by platelet aggregation in response to various agonists, platelet deposition under flow conditions in the Impact-R system, and plasma levels of platelet activation markers (soluble CD40 ligand and P-selectin). In the OPVT group, the average INR during the index episode was 3.1 ⴞ 1.5, and 44.6 ⴞ 40 months had elapsed from the index episode to the present study. Both groups were matched for gender, age ⴞ10 years, valve position and type, active smoking, and diabetes. Patients with an OPVT history had a greater aggregation in response to collagen (p ⴝ 0.05), greater platelet deposition in the Impact-R system (p ⴝ 0.01), and tended to have higher levels of soluble P-selection and soluble CD40 ligand (p ⴝ 0.07) than their control counterparts. In conclusion, patients with a history of OPVT appear to have increased platelet reactivity, which might contribute to an increased risk of thrombotic complications. These patients would, therefore, likely benefit from the addition of antiplatelet therapy to their standard anticoagulant treatment. © 2009 Elsevier Inc. (Am J Cardiol 2009;103:1760 –1763)
One of the most serious complications of a prosthetic mechanical valve is the occurrence of obstructive prosthetic valve thrombosis (OPVT), also referred to as a “stuck” mechanical valve. The development of an OPVT episode can be caused by a blood clot, pannus growth, or a combination of the 2.1 From our experience at the Rabin Medical Center, Israel, in ⬎80% of patients who presented with OPVT, valve thrombosis was resolved by administering thrombolytic therapy, suggesting the involvement of a fresh blood clot.2 Some patients develop this complication despite the optimal use of warfarin and a therapeutic international normalized ratio (INR) at presentation. The development of an obstructive thrombus despite “effective” anticoagulant treatment suggests that activated platelets might also have a role in the pathogenesis of the clot. We hypothesized that patients who develop an episode of OPVT have hyperreactive platelets, contributing to their propensity to develop thrombotic complications. Accordingly, our aim was to examine platelet reactivity in patients who developed OPVT, despite a therapeutic or near-therapeutic INR, compared
Department of Cardiology, Rabin Medical Center, Petah-Tikva, affiliated with Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. *Corresponding author: Tel: 9723-9376431; fax: 9723-9231016. E-mail address: [email protected] (E.I. Lev). 0002-9149/09/$ – see front matter © 2009 Elsevier Inc. doi:10.1016/j.amjcard.2009.02.031
with a matched group of patients with mechanical valves who never developed this complication. Methods The present study was a retrospective case-control study. We identified patients who had had an episode of OPVT of a mechanical valve from 1996 to 2007 and were hospitalized at the Rabin Medical Center, Israel (a tertiary medical center). The inclusion criteria for participation in the study were ⱖ1 episode of OPVT; a recorded INR at a therapeutic or near-therapeutic level at the admission for OPVT (we defined near-therapeutic as ⱕ0.5 below the low range recommended INR according to the American Heart Association guidelines for the specific mechanical valve1); ⱖ3 months had elapsed from the index episode (to avoid platelet hyper-reactivity secondary to the acute episode); and the patient was available for follow-up. The use of aspirin at the acute episode was not an exclusion criterion. A control patient was matched to each patient in the study group. The control group consisted of patients with mechanical valves who had never experienced OPVT. They were matched to the study group patients according to the following criteria: gender, age ⫾10 years, valve position and type, anticoagulation and antiplatelet treatment at the study (e.g., warfarin and low-dose aspirin); active smoking, and the presence or absence of diabetes mellitus. The exclusion criteria for both groups were thrombocytopenia www.AJConline.org
Valvular Heart Disease/Platelet Function in Patients With OVPD
(⬍100 ⫻ 103 cells/mm3) and anemia (hemoglobin ⬍10 g/dL). The investigational review board (ethics committee) of the Rabin Medical Center, Israel, approved the study, and all subjects provided written informed consent. In both groups, 1 blood sample was taken after the patient had from midnight and in a resting state. The sample was drawn from an antecubital vein using a 19-gauge needle. For each sample, the blood was collected in tubes containing 3.2% citrate. The tubes were filled to capacity and then gently mixed. The blood samples were processed within 1 hour of blood collection and evaluated by platelet aggregation and the Impact-R system (DiaMed, Cresier, Switzerland). Turbidimetric platelet aggregation was performed in platelet-rich plasma with a platelet count adjusted to 250 ⫻ 103/mm3. The platelets were stimulated with 0.5 mg/ml (⬃1.5 mM) arachidonic acid, 5 and 10 M adenosine diphosphate (ADP), and 1 g/ml collagen. Aggregation was performed with a BioData PAP-4 platelet aggregometer (BioData, Horsham, Pennsylvania). Platelet-poor plasma was used as a reference. The extent of aggregation was defined according to 2 parameters: the maximal amount of light transmission reached at ⱕ6 minutes after addition of the agonist, and the extent of aggregation at 6 minutes. The Impact-R device is based on the cone and plate(let) analyzer technology and evaluates platelet deposition under high shear rate conditions.3 Platelet deposition under flow is dependent on platelet activation and the presence of fibrinogen, von Willebrand factor, and their receptors.3 In brief, the whole blood sample (130 l) was placed in a polystyrene well and subjected to flow (1,800 s⫺1 for 2 minutes) using a rotating cone. The well was washed and stained with May-Gruenwald stain. Platelet deposition was evaluated using the Impact-R image analysis system. The results are expressed as the percentage of surface coverage (reflecting platelet adhesion), and the average aggregate size (reflecting aggregate formation). Plasma levels of soluble P-selectin and soluble CD40 ligand were measured from plasma stored at ⫺70°C, in duplicate, using an enzyme-linked immunosorbent assay, according to the manufacturer’s instructions and using commercial reagents and recombinant human antibodies as standard (R&D Systems, Minneapolis, Minnesota). Continuous variables are presented as the mean ⫾ standard deviation. Comparison of continuous variables between the 2 groups was performed using Student’s unpaired t tests, because the distribution of the platelet function tests in both groups appeared to be normal according to the Kolmogorov-Smirnov test. Subgroup (n ⬍12) comparisons were performed using the nonparametric Wilcoxon ranksum test. Categorical variables were compared using chisquare tests. Analyses were performed using Statistical Package for Social Sciences, version 11.0, statistical software (SPSS, Chicago, Illinois), and statistical significance was set at p ⬍0.05. Results The study group consisted of 18 patients with an episode of OPVT from 1997 to 2007. All the valves were bileaflet mechanical valves. Of the 18 patients, 9 (50%) had mechan-
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Table 1 Clinical characteristics Variable
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
Age (yrs) Women Warfarin* Low-dose aspirin* Current smoking Diabetes mellitus Mechanical bileaflet valve Mitral position Aortic position Tricuspid position Interval from valve implantation (yrs) Previous stroke Chronic/paroxysmal atrial fibrillation Ischemic heart disease Dyslipidemia† Hypertension† Platelet count (103/l) Lactic dehydrogenase (U/L)
56.3 ⫾ 14 11 (61%) 18 (100%) 10 (58%) 2 (11%) 3 (17%) 18 (100%) 9 (50%) 6 (33%) 3 (17%) 6.8 ⫾ 4 5 (28%) 11 (61%) 8 (44%) 12 (68%) 13 (72%) 320 ⫾ 117 533 ⫾ 96
58.2 ⫾ 15 11 (61%) 18 (100%) 10 (58%) 2 (11%) 3 (17%) 18 (100%) 9 (50%) 6 (33%) 3 (17%) 5.3 ⫾ 3 2 (11%) 10 (56%) 5 (28%) 9 (50%) 10 (56%) 281 ⫾ 98 552 ⫾ 97
* Present treatment. † Hyperlipidemia/hypertension— diagnosis previously made by physician or patient receiving lipid-lowering therapy.
ical valves at the mitral position, 6 (33.3%) at the aortic position, and 3 (16.7%) at the tricuspid location. The average admission INR at the OPVT episode (index episode) was 3.1 ⫾ 1.5. The average period elapsed from the index OPVT episode to the present study was 44.6 ⫾ 40 months. All patients were treated acutely with thrombolysis. After the OPVT index episode and before the present study, 8 patients (44.4%) had undergone valve replacement surgery (all received another bileaflet mechanical valve). The control group consisted of 18 matched patients with mechanical valves who had not experienced an OPVT episode. The mean INR at testing was 2.9 ⫾ 0.7 in the study group and 2.8 ⫾ 0.9 in the control group (p ⫽ NS). The clinical characteristics of the 2 groups are presented in Table 1. No significant differences were found between the groups in any of the parameters. In both groups, all patients were treated with warfarin and 55.6% with aspirin (100 mg daily). None of the patients were taking clopidogrel. Table 2 lists platelet aggregation (light transmission aggregation) in response to various agonists. Aggregation in response to ADP and arachidonic acid did not differ between the 2 groups. However, aggregation in response to collagen was greater in the study group than in the control group (p ⫽ 0.04 to 0.05 for 6-minute and maximal aggregation). When the groups were analyzed according to the subgroups of patients who were or were not taking aspirin, the results were similar to those for the whole group (Table 3). Platelet deposition under flow conditions, measured as the platelet surface coverage and average aggregate size in the Impact-R system, was greater in the study group than in the control group (p ⫽ 0.01 to 0.04 for surface coverage and aggregate size; Table 4). In addition, patients in the study group tended to have greater levels of platelet activation markers—soluble P-selectin and soluble CD40 ligand— than their control counterparts (p ⫽ 0.07; Table 4).
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Table 2 Platelet aggregation in study vs control groups Agonist
ADP (mol/L) 5 10 AA 0.5 mg/ml Collagen 1 g/ml
6-Minute Aggregation (%)
Maximal aggregation (%)
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
p Value
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
p Value
59.6 ⫾ 16 68.2 ⫾ 13 40 ⫾ 33 66.9 ⫾ 24
57.1 ⫾ 16 65.5 ⫾ 15 34.8 ⫾ 33 48.4 ⫾ 28
0.7 0.6 0.6 0.04
61.8 ⫾ 14 69.3 ⫾ 13 41.2 ⫾ 32 69.7 ⫾ 25
60.2 ⫾ 14 67 ⫾ 13 35.6 ⫾ 33 51.8 ⫾ 29
0.8 0.7 0.6 0.05
AA ⫽ arachidonic acid.
Table 3 Maximal platelet aggregation according to aspirin treatment Agonist
ADP (mol/L) 5 10 AA 0.5 mg/ml Collagen 1 g/ml
Taking Aspirin
Not Taking Aspirin
Study Group (n ⫽ 10)
Control Group (n ⫽ 10)
p Value
Study Group (n ⫽ 8)
Control Group (n ⫽ 8)
p Value
54.9 ⫾ 9 62.7 ⫾ 7 12.4 ⫾ 7 60.7 ⫾ 25
54 ⫾ 10 63 ⫾ 8 10.8 ⫾ 6 44 ⫾ 28
0.9 1 0.5 0.15
70.4 ⫾ 15 77.6 ⫾ 14 77.5 ⫾ 12 80.7 ⫾ 8
64.9 ⫾ 17 71.5 ⫾ 20 67.4 ⫾ 20 61.4 ⫾ 21
0.5 0.6 0.4 0.06
AA ⫽ arachidonic acid. Table 4 Platelet reactivity tests in study vs control groups Test
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
p Value
Impact-R–SC (%) Impact-R–AS (m2) Soluble CD40 ligand (pg/ml) Soluble P-selectin (ng/ml)
9.1 ⫾ 3.4 44.6 ⫾ 11 201.7 ⫾ 205 3.0 ⫾ 2.3
6.5 ⫾ 2.5 36.7 ⫾ 10 63.5 ⫾ 62 1.9 ⫾ 0.4
0.01 0.04 0.07 0.07
SC ⫽ surface coverage; AS ⫽ average size.
Discussion This is the first study to examine platelet reactivity in patients who had had an episode of OPVT of a mechanical valve compared with the platelet reactivity in a wellmatched control group. Platelet function was assessed by a variety of assays, reflecting the various aspects of platelet reactivity. In this case-control study, we found that patients with a history of OPVT had increased platelet reactivity as evaluated by light transmission aggregation in response to collagen, platelet deposition under flow conditions in the Impact-R system, and a trend for greater platelet activation marker levels—soluble P-selectin and soluble CD40 ligand. These assays reflect the platelet activation state, granule release in response to activation, and aggregation and/or adhesion in response to chemical stimuli (collagen) or shear rate. The lack of a difference in the arachidonic acid- and ADP-induced platelet aggregation might have been because these agonists are more “pathway specific” in their stimulatory effects, and the resulting aggregation is affected by aspirin, thus creating 2 small subgroups (with or without aspirin treatment) in each group. Overall, our findings suggest that platelet hyper-reactivity, observed in the patients
who had had an OPVT episode, might have contributed to the development of the thrombotic complication. The process of thrombus formation on a prosthetic mechanical heart valve appears to involve 2 concurrent mechanisms.4 The first is disruption of the vascular endothelial surface and exposure of the underlying prothrombotic substrate. The second mechanism is dependent on the flow rate. In areas with rapid blood flow and high shear stress (e.g., flow through an aortic valve), platelets are activated and erythrocyte membranes are damaged. The latter process leads to erythrocyte lysis and the release of ADP—a potent platelet agonist. Thus, erythrocyte fragmentation and platelet activation contribute to thrombus formation in this scenario. In contrast, in areas of slow flow and stasis (e.g., a mitral valve, particularly with left atrial enlargement), contact of coagulation factors with the damaged prosthetic surface is prolonged. In this setting, coagulation factor activation is probably the predominant process for thrombus formation, and platelet recruitment represents an essential, but secondary, step. The extension of thrombus into regions of relative stasis is common in both arterial and venous thromboembolism and represents an important mechanism for thrombus growth after prosthetic heart valve surgery.4 Artificial surfaces such as prosthetic mechanical valves can promote platelet activation.4 – 6 This process is dependent on the surface properties, including physical characteristics, electrical charge, and surface chemistry, and on the shear rate of blood flow.4 Therefore, the mechanical valve itself might have influenced the platelet reactivity indexes measured in our study. However, because both our study and control group patients had mechanical valves matched by their location and type, the differences in platelet reactivity observed in our study imply that the group of patients
Valvular Heart Disease/Platelet Function in Patients With OVPD
who had had an OPVT episode had inherently increased platelet reactivity (independent of, or in addition to, that induced by the surface of the mechanical valves). Although previous studies have not directly examined platelet reactivity in patients who developed OPVT, a few studies have demonstrated the importance of antiplatelet therapy in preventing thrombus formation in patients with mechanical valves. Schlitt et al7 reported that in an ex vivo rabbit model, combined therapy with clopidogrel and aspirin was more effective than warfarin in preventing thrombus formation on artificial heart valve leaflets. Furthermore, Laffort et al8 showed that the addition of aspirin to anticoagulant therapy for patients after mechanical mitral valve replacement was associated with a lower incidence of nonobstructive periprosthetic valve thrombi, as detected by transesophageal echocardiography. In accordance with these findings, several large clinical studies and meta-analyses have demonstrated that in patients with mechanical heart valves and high-risk patients with prosthetic tissue valves, the addition of aspirin to warfarin therapy reduced the risk of thromboembolic events, as well as total mortality, and the risk of significant bleeding was only mildly elevated.9 –12 The present study had several limitations. First, the sample size was relatively small (n ⫽ 18). However, despite the small size, we were able to demonstrate significant or nearsignificant differences in most platelet assays, emphasizing the unique platelet profile in this patient group. Second, we only recruited patients who had survived the OPVT episode and were available for follow-up, possibly creating a selection bias. Third, although we intended to select patients with therapeutic or near-therapeutic INR levels at the OPVT episode, we only had data regarding the INR at the hospital admission with the complication. Previous INR values (before presentation to the hospital) might have also been relevant to the development of the thrombotic complication. Fourth, despite the matching process, residual confounders might have been present. Finally, platelet reactivity was measured at a single point—months to years after the OPVT episode and not before its development. Thus, it is impossible to determine with certainty that platelet hyper-reactivity was involved in the pathogenesis of the index acute episode. However, we measured platelet reactivity at a “resting” state, a substantial period after the acute event, which might itself have induced secondary platelet activation. Although fluctuations in platelet reactivity are possible, we assumed this measurement reflects the basal properties of the platelets in the examined patient. In conclusion,
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our findings of elevated platelet reactivity in patients who developed an episode of OPVT lend support to the hypothesis that platelet hyper-reactivity might contribute to the development of a thrombotic process leading to valve obstruction. 1. Bonow RO, Carabello BA, Chatterjee K, de Leon AC Jr, Faxon DP, Freed MD, Gaasch WH, Lytle BW, Nishimura RA, O’Gara PT, O’Rourke RA, Otto CM, Shah PM, Shanewise JS; American College of Cardiology/American Heart Association Task Force on Practice Guidelines. 2008 Focused update incorporated into the ACC/AHA 2006 guidelines for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice guidelines. J Am Coll Cardiol 2008;52:1–142. 2. Shapira Y, Herz I, Vaturi M, Porter A, Adler Y, Birnbaum Y, Strasberg B, Sclarovsky S, Sagie A. Thrombolysis is an effective and safe therapy in stuck bileaflet mitral valves in the absence of high-risk thrombi. J Am Coll Cardiol 2000;35:1874 –1880. 3. Shenkman B, Savion N, Dardik R, Tamarin I, Varon D. Testing of platelet deposition on polystyrene surface under flow conditions by the cone and plate(let) analyzer: role of platelet activation, fibrinogen and von Willebrand factor. Thromb Res 2000;99:353–361. 4. Becker RC, Eisenberg P, Turpie AG. Pathobiologic features and prevention of thrombotic complications associated with prosthetic heart valves: fundamental principles and the contribution of platelets and thrombin. Am Heart J 2001;141:1025–1037. 5. Courtney JM, Forbes CD. Thrombosis on foreign surfaces. Br Med Bull 1994;50:966 –981. 6. Forbes CD. Thrombosis and artificial surfaces. Clin Haematol 1981; 10:653– 668. 7. Schlitt A, Hauroeder B, Buerke M, Peetz D, Victor A, Hundt F, Bickel C, Meyer J, Rupprecht HJ. Effects of combined therapy of clopidogrel and aspirin in preventing thrombus formation on mechanical heart valves in an ex vivo rabbit model. Thromb Res 2002;107:39 – 43. 8. Laffort P, Roudaut R, Roques X, Lafitte S, Deville C, Bonnet J, Baudet E. Early and long-term (one-year) effects of the association of aspirin and oral anticoagulant on thrombi and morbidity after replacement of the mitral valve with the St. Jude Medical prosthesis: a clinical and transesophageal echocardiographic study. J Am Coll Cardiol 2000;35: 739 –746. 9. Turpie AG, Gent M, Laupacis A, Latour Y, Gunstensen J, Basile F, Klimek M, Hirsh J. A comparison of aspirin with placebo in patients treated with warfarin after heart-valve replacement. N Engl J Med 1993;329:524 –529. 10. Cappelleri JC, Fiore LD, Brophy MT, Deykin D, Lau J. Efficacy and safety of combined anticoagulant and antiplatelet therapy versus anticoagulant monotherapy after mechanical heart-valve replacement: a meta-analysis. Am Heart J 1995;130:547–552. 11. Massel D, Little SH. Risks and benefits of adding anti-platelet therapy to warfarin among patients with prosthetic heart valves: a meta-analysis. J Am Coll Cardiol 2001;37:869 – 878. 12. Meschengieser SS, Fondevila CG, Frontroth J, Sandarelli MT, Lazzari MA. Low-intensity oral anticoagulation plus low dose aspirin versus high-intensity oral anticoagulation alone: a randomized trial in patients with mechanical heart valves. J Thorac Cardiovasc Surg 1997;113: 910 –916.
One of the most serious complications of a prosthetic mechanical valve is the occurrence of obstructive prosthetic valve thrombosis (OPVT), also referred to as a “stuck” mechanical valve. The development of an OPVT episode can be caused by a blood clot, pannus growth, or a combination of the 2.1 From our experience at the Rabin Medical Center, Israel, in ⬎80% of patients who presented with OPVT, valve thrombosis was resolved by administering thrombolytic therapy, suggesting the involvement of a fresh blood clot.2 Some patients develop this complication despite the optimal use of warfarin and a therapeutic international normalized ratio (INR) at presentation. The development of an obstructive thrombus despite “effective” anticoagulant treatment suggests that activated platelets might also have a role in the pathogenesis of the clot. We hypothesized that patients who develop an episode of OPVT have hyperreactive platelets, contributing to their propensity to develop thrombotic complications. Accordingly, our aim was to examine platelet reactivity in patients who developed OPVT, despite a therapeutic or near-therapeutic INR, compared
Department of Cardiology, Rabin Medical Center, Petah-Tikva, affiliated with Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. *Corresponding author: Tel: 9723-9376431; fax: 9723-9231016. E-mail address: [email protected] (E.I. Lev). 0002-9149/09/$ – see front matter © 2009 Elsevier Inc. doi:10.1016/j.amjcard.2009.02.031
with a matched group of patients with mechanical valves who never developed this complication. Methods The present study was a retrospective case-control study. We identified patients who had had an episode of OPVT of a mechanical valve from 1996 to 2007 and were hospitalized at the Rabin Medical Center, Israel (a tertiary medical center). The inclusion criteria for participation in the study were ⱖ1 episode of OPVT; a recorded INR at a therapeutic or near-therapeutic level at the admission for OPVT (we defined near-therapeutic as ⱕ0.5 below the low range recommended INR according to the American Heart Association guidelines for the specific mechanical valve1); ⱖ3 months had elapsed from the index episode (to avoid platelet hyper-reactivity secondary to the acute episode); and the patient was available for follow-up. The use of aspirin at the acute episode was not an exclusion criterion. A control patient was matched to each patient in the study group. The control group consisted of patients with mechanical valves who had never experienced OPVT. They were matched to the study group patients according to the following criteria: gender, age ⫾10 years, valve position and type, anticoagulation and antiplatelet treatment at the study (e.g., warfarin and low-dose aspirin); active smoking, and the presence or absence of diabetes mellitus. The exclusion criteria for both groups were thrombocytopenia www.AJConline.org
Valvular Heart Disease/Platelet Function in Patients With OVPD
(⬍100 ⫻ 103 cells/mm3) and anemia (hemoglobin ⬍10 g/dL). The investigational review board (ethics committee) of the Rabin Medical Center, Israel, approved the study, and all subjects provided written informed consent. In both groups, 1 blood sample was taken after the patient had from midnight and in a resting state. The sample was drawn from an antecubital vein using a 19-gauge needle. For each sample, the blood was collected in tubes containing 3.2% citrate. The tubes were filled to capacity and then gently mixed. The blood samples were processed within 1 hour of blood collection and evaluated by platelet aggregation and the Impact-R system (DiaMed, Cresier, Switzerland). Turbidimetric platelet aggregation was performed in platelet-rich plasma with a platelet count adjusted to 250 ⫻ 103/mm3. The platelets were stimulated with 0.5 mg/ml (⬃1.5 mM) arachidonic acid, 5 and 10 M adenosine diphosphate (ADP), and 1 g/ml collagen. Aggregation was performed with a BioData PAP-4 platelet aggregometer (BioData, Horsham, Pennsylvania). Platelet-poor plasma was used as a reference. The extent of aggregation was defined according to 2 parameters: the maximal amount of light transmission reached at ⱕ6 minutes after addition of the agonist, and the extent of aggregation at 6 minutes. The Impact-R device is based on the cone and plate(let) analyzer technology and evaluates platelet deposition under high shear rate conditions.3 Platelet deposition under flow is dependent on platelet activation and the presence of fibrinogen, von Willebrand factor, and their receptors.3 In brief, the whole blood sample (130 l) was placed in a polystyrene well and subjected to flow (1,800 s⫺1 for 2 minutes) using a rotating cone. The well was washed and stained with May-Gruenwald stain. Platelet deposition was evaluated using the Impact-R image analysis system. The results are expressed as the percentage of surface coverage (reflecting platelet adhesion), and the average aggregate size (reflecting aggregate formation). Plasma levels of soluble P-selectin and soluble CD40 ligand were measured from plasma stored at ⫺70°C, in duplicate, using an enzyme-linked immunosorbent assay, according to the manufacturer’s instructions and using commercial reagents and recombinant human antibodies as standard (R&D Systems, Minneapolis, Minnesota). Continuous variables are presented as the mean ⫾ standard deviation. Comparison of continuous variables between the 2 groups was performed using Student’s unpaired t tests, because the distribution of the platelet function tests in both groups appeared to be normal according to the Kolmogorov-Smirnov test. Subgroup (n ⬍12) comparisons were performed using the nonparametric Wilcoxon ranksum test. Categorical variables were compared using chisquare tests. Analyses were performed using Statistical Package for Social Sciences, version 11.0, statistical software (SPSS, Chicago, Illinois), and statistical significance was set at p ⬍0.05. Results The study group consisted of 18 patients with an episode of OPVT from 1997 to 2007. All the valves were bileaflet mechanical valves. Of the 18 patients, 9 (50%) had mechan-
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Table 1 Clinical characteristics Variable
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
Age (yrs) Women Warfarin* Low-dose aspirin* Current smoking Diabetes mellitus Mechanical bileaflet valve Mitral position Aortic position Tricuspid position Interval from valve implantation (yrs) Previous stroke Chronic/paroxysmal atrial fibrillation Ischemic heart disease Dyslipidemia† Hypertension† Platelet count (103/l) Lactic dehydrogenase (U/L)
56.3 ⫾ 14 11 (61%) 18 (100%) 10 (58%) 2 (11%) 3 (17%) 18 (100%) 9 (50%) 6 (33%) 3 (17%) 6.8 ⫾ 4 5 (28%) 11 (61%) 8 (44%) 12 (68%) 13 (72%) 320 ⫾ 117 533 ⫾ 96
58.2 ⫾ 15 11 (61%) 18 (100%) 10 (58%) 2 (11%) 3 (17%) 18 (100%) 9 (50%) 6 (33%) 3 (17%) 5.3 ⫾ 3 2 (11%) 10 (56%) 5 (28%) 9 (50%) 10 (56%) 281 ⫾ 98 552 ⫾ 97
* Present treatment. † Hyperlipidemia/hypertension— diagnosis previously made by physician or patient receiving lipid-lowering therapy.
ical valves at the mitral position, 6 (33.3%) at the aortic position, and 3 (16.7%) at the tricuspid location. The average admission INR at the OPVT episode (index episode) was 3.1 ⫾ 1.5. The average period elapsed from the index OPVT episode to the present study was 44.6 ⫾ 40 months. All patients were treated acutely with thrombolysis. After the OPVT index episode and before the present study, 8 patients (44.4%) had undergone valve replacement surgery (all received another bileaflet mechanical valve). The control group consisted of 18 matched patients with mechanical valves who had not experienced an OPVT episode. The mean INR at testing was 2.9 ⫾ 0.7 in the study group and 2.8 ⫾ 0.9 in the control group (p ⫽ NS). The clinical characteristics of the 2 groups are presented in Table 1. No significant differences were found between the groups in any of the parameters. In both groups, all patients were treated with warfarin and 55.6% with aspirin (100 mg daily). None of the patients were taking clopidogrel. Table 2 lists platelet aggregation (light transmission aggregation) in response to various agonists. Aggregation in response to ADP and arachidonic acid did not differ between the 2 groups. However, aggregation in response to collagen was greater in the study group than in the control group (p ⫽ 0.04 to 0.05 for 6-minute and maximal aggregation). When the groups were analyzed according to the subgroups of patients who were or were not taking aspirin, the results were similar to those for the whole group (Table 3). Platelet deposition under flow conditions, measured as the platelet surface coverage and average aggregate size in the Impact-R system, was greater in the study group than in the control group (p ⫽ 0.01 to 0.04 for surface coverage and aggregate size; Table 4). In addition, patients in the study group tended to have greater levels of platelet activation markers—soluble P-selectin and soluble CD40 ligand— than their control counterparts (p ⫽ 0.07; Table 4).
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Table 2 Platelet aggregation in study vs control groups Agonist
ADP (mol/L) 5 10 AA 0.5 mg/ml Collagen 1 g/ml
6-Minute Aggregation (%)
Maximal aggregation (%)
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
p Value
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
p Value
59.6 ⫾ 16 68.2 ⫾ 13 40 ⫾ 33 66.9 ⫾ 24
57.1 ⫾ 16 65.5 ⫾ 15 34.8 ⫾ 33 48.4 ⫾ 28
0.7 0.6 0.6 0.04
61.8 ⫾ 14 69.3 ⫾ 13 41.2 ⫾ 32 69.7 ⫾ 25
60.2 ⫾ 14 67 ⫾ 13 35.6 ⫾ 33 51.8 ⫾ 29
0.8 0.7 0.6 0.05
AA ⫽ arachidonic acid.
Table 3 Maximal platelet aggregation according to aspirin treatment Agonist
ADP (mol/L) 5 10 AA 0.5 mg/ml Collagen 1 g/ml
Taking Aspirin
Not Taking Aspirin
Study Group (n ⫽ 10)
Control Group (n ⫽ 10)
p Value
Study Group (n ⫽ 8)
Control Group (n ⫽ 8)
p Value
54.9 ⫾ 9 62.7 ⫾ 7 12.4 ⫾ 7 60.7 ⫾ 25
54 ⫾ 10 63 ⫾ 8 10.8 ⫾ 6 44 ⫾ 28
0.9 1 0.5 0.15
70.4 ⫾ 15 77.6 ⫾ 14 77.5 ⫾ 12 80.7 ⫾ 8
64.9 ⫾ 17 71.5 ⫾ 20 67.4 ⫾ 20 61.4 ⫾ 21
0.5 0.6 0.4 0.06
AA ⫽ arachidonic acid. Table 4 Platelet reactivity tests in study vs control groups Test
Study Group (n ⫽ 18)
Control Group (n ⫽ 18)
p Value
Impact-R–SC (%) Impact-R–AS (m2) Soluble CD40 ligand (pg/ml) Soluble P-selectin (ng/ml)
9.1 ⫾ 3.4 44.6 ⫾ 11 201.7 ⫾ 205 3.0 ⫾ 2.3
6.5 ⫾ 2.5 36.7 ⫾ 10 63.5 ⫾ 62 1.9 ⫾ 0.4
0.01 0.04 0.07 0.07
SC ⫽ surface coverage; AS ⫽ average size.
Discussion This is the first study to examine platelet reactivity in patients who had had an episode of OPVT of a mechanical valve compared with the platelet reactivity in a wellmatched control group. Platelet function was assessed by a variety of assays, reflecting the various aspects of platelet reactivity. In this case-control study, we found that patients with a history of OPVT had increased platelet reactivity as evaluated by light transmission aggregation in response to collagen, platelet deposition under flow conditions in the Impact-R system, and a trend for greater platelet activation marker levels—soluble P-selectin and soluble CD40 ligand. These assays reflect the platelet activation state, granule release in response to activation, and aggregation and/or adhesion in response to chemical stimuli (collagen) or shear rate. The lack of a difference in the arachidonic acid- and ADP-induced platelet aggregation might have been because these agonists are more “pathway specific” in their stimulatory effects, and the resulting aggregation is affected by aspirin, thus creating 2 small subgroups (with or without aspirin treatment) in each group. Overall, our findings suggest that platelet hyper-reactivity, observed in the patients
who had had an OPVT episode, might have contributed to the development of the thrombotic complication. The process of thrombus formation on a prosthetic mechanical heart valve appears to involve 2 concurrent mechanisms.4 The first is disruption of the vascular endothelial surface and exposure of the underlying prothrombotic substrate. The second mechanism is dependent on the flow rate. In areas with rapid blood flow and high shear stress (e.g., flow through an aortic valve), platelets are activated and erythrocyte membranes are damaged. The latter process leads to erythrocyte lysis and the release of ADP—a potent platelet agonist. Thus, erythrocyte fragmentation and platelet activation contribute to thrombus formation in this scenario. In contrast, in areas of slow flow and stasis (e.g., a mitral valve, particularly with left atrial enlargement), contact of coagulation factors with the damaged prosthetic surface is prolonged. In this setting, coagulation factor activation is probably the predominant process for thrombus formation, and platelet recruitment represents an essential, but secondary, step. The extension of thrombus into regions of relative stasis is common in both arterial and venous thromboembolism and represents an important mechanism for thrombus growth after prosthetic heart valve surgery.4 Artificial surfaces such as prosthetic mechanical valves can promote platelet activation.4 – 6 This process is dependent on the surface properties, including physical characteristics, electrical charge, and surface chemistry, and on the shear rate of blood flow.4 Therefore, the mechanical valve itself might have influenced the platelet reactivity indexes measured in our study. However, because both our study and control group patients had mechanical valves matched by their location and type, the differences in platelet reactivity observed in our study imply that the group of patients
Valvular Heart Disease/Platelet Function in Patients With OVPD
who had had an OPVT episode had inherently increased platelet reactivity (independent of, or in addition to, that induced by the surface of the mechanical valves). Although previous studies have not directly examined platelet reactivity in patients who developed OPVT, a few studies have demonstrated the importance of antiplatelet therapy in preventing thrombus formation in patients with mechanical valves. Schlitt et al7 reported that in an ex vivo rabbit model, combined therapy with clopidogrel and aspirin was more effective than warfarin in preventing thrombus formation on artificial heart valve leaflets. Furthermore, Laffort et al8 showed that the addition of aspirin to anticoagulant therapy for patients after mechanical mitral valve replacement was associated with a lower incidence of nonobstructive periprosthetic valve thrombi, as detected by transesophageal echocardiography. In accordance with these findings, several large clinical studies and meta-analyses have demonstrated that in patients with mechanical heart valves and high-risk patients with prosthetic tissue valves, the addition of aspirin to warfarin therapy reduced the risk of thromboembolic events, as well as total mortality, and the risk of significant bleeding was only mildly elevated.9 –12 The present study had several limitations. First, the sample size was relatively small (n ⫽ 18). However, despite the small size, we were able to demonstrate significant or nearsignificant differences in most platelet assays, emphasizing the unique platelet profile in this patient group. Second, we only recruited patients who had survived the OPVT episode and were available for follow-up, possibly creating a selection bias. Third, although we intended to select patients with therapeutic or near-therapeutic INR levels at the OPVT episode, we only had data regarding the INR at the hospital admission with the complication. Previous INR values (before presentation to the hospital) might have also been relevant to the development of the thrombotic complication. Fourth, despite the matching process, residual confounders might have been present. Finally, platelet reactivity was measured at a single point—months to years after the OPVT episode and not before its development. Thus, it is impossible to determine with certainty that platelet hyper-reactivity was involved in the pathogenesis of the index acute episode. However, we measured platelet reactivity at a “resting” state, a substantial period after the acute event, which might itself have induced secondary platelet activation. Although fluctuations in platelet reactivity are possible, we assumed this measurement reflects the basal properties of the platelets in the examined patient. In conclusion,
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our findings of elevated platelet reactivity in patients who developed an episode of OPVT lend support to the hypothesis that platelet hyper-reactivity might contribute to the development of a thrombotic process leading to valve obstruction. 1. Bonow RO, Carabello BA, Chatterjee K, de Leon AC Jr, Faxon DP, Freed MD, Gaasch WH, Lytle BW, Nishimura RA, O’Gara PT, O’Rourke RA, Otto CM, Shah PM, Shanewise JS; American College of Cardiology/American Heart Association Task Force on Practice Guidelines. 2008 Focused update incorporated into the ACC/AHA 2006 guidelines for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice guidelines. J Am Coll Cardiol 2008;52:1–142. 2. Shapira Y, Herz I, Vaturi M, Porter A, Adler Y, Birnbaum Y, Strasberg B, Sclarovsky S, Sagie A. Thrombolysis is an effective and safe therapy in stuck bileaflet mitral valves in the absence of high-risk thrombi. J Am Coll Cardiol 2000;35:1874 –1880. 3. Shenkman B, Savion N, Dardik R, Tamarin I, Varon D. Testing of platelet deposition on polystyrene surface under flow conditions by the cone and plate(let) analyzer: role of platelet activation, fibrinogen and von Willebrand factor. Thromb Res 2000;99:353–361. 4. Becker RC, Eisenberg P, Turpie AG. Pathobiologic features and prevention of thrombotic complications associated with prosthetic heart valves: fundamental principles and the contribution of platelets and thrombin. Am Heart J 2001;141:1025–1037. 5. Courtney JM, Forbes CD. Thrombosis on foreign surfaces. Br Med Bull 1994;50:966 –981. 6. Forbes CD. Thrombosis and artificial surfaces. Clin Haematol 1981; 10:653– 668. 7. Schlitt A, Hauroeder B, Buerke M, Peetz D, Victor A, Hundt F, Bickel C, Meyer J, Rupprecht HJ. Effects of combined therapy of clopidogrel and aspirin in preventing thrombus formation on mechanical heart valves in an ex vivo rabbit model. Thromb Res 2002;107:39 – 43. 8. Laffort P, Roudaut R, Roques X, Lafitte S, Deville C, Bonnet J, Baudet E. Early and long-term (one-year) effects of the association of aspirin and oral anticoagulant on thrombi and morbidity after replacement of the mitral valve with the St. Jude Medical prosthesis: a clinical and transesophageal echocardiographic study. J Am Coll Cardiol 2000;35: 739 –746. 9. Turpie AG, Gent M, Laupacis A, Latour Y, Gunstensen J, Basile F, Klimek M, Hirsh J. A comparison of aspirin with placebo in patients treated with warfarin after heart-valve replacement. N Engl J Med 1993;329:524 –529. 10. Cappelleri JC, Fiore LD, Brophy MT, Deykin D, Lau J. Efficacy and safety of combined anticoagulant and antiplatelet therapy versus anticoagulant monotherapy after mechanical heart-valve replacement: a meta-analysis. Am Heart J 1995;130:547–552. 11. Massel D, Little SH. Risks and benefits of adding anti-platelet therapy to warfarin among patients with prosthetic heart valves: a meta-analysis. J Am Coll Cardiol 2001;37:869 – 878. 12. Meschengieser SS, Fondevila CG, Frontroth J, Sandarelli MT, Lazzari MA. Low-intensity oral anticoagulation plus low dose aspirin versus high-intensity oral anticoagulation alone: a randomized trial in patients with mechanical heart valves. J Thorac Cardiovasc Surg 1997;113: 910 –916.