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a Division of Cardiothoracic and Vascular Anesthesia and Intensive Care, Medical University of Vienna, Vienna, Austria
b Division of Cardiothoracic Surgery, Medical University of Vienna, Vienna, Austria
c Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria
d Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria
Accepted for publication October 14, 2008.
* Address correspondence to Dr Steinlechner, Division of Cardiothoracic and Vascular Anesthesia and Intensive Care, Medical University of Vienna, Waehringer Guertel 18-20, Vienna, A-1090, Austria (Email: barbara.steinlechner{at}meduniwien.ac.at).
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Abstract |
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Methods: In a cross-sectional design platelet function was assessed in 12 outpatients and 12 healthy matched volunteers using thrombelastography platelet mapping, thromboelastometry, platelet function analyzer, and a new whole blood aggregometer (Multiplate).
Results: Phenprocoumon produced an international normalized ratio of 3.5. It was associated with a twofold prolongation in the thromboelastometry clotting time (p < 0.001). Platelet function under high shear was severely compromised: collagen adenosine diphosphate closure times were 2.5-fold longer in patients than in volunteers (p < 0.001), and 50% of patients had maximal collagen adenosine diphosphate closure time values. Although antigen levels of von Willebrand factor were 80% higher in patients (p < 0.001), von Willebrand factor–ristocetin was subnormal in 5 of 12 patients. Ristocetin-induced aggregation was also threefold higher in volunteers (p < 0.001), indicating an additional functional defect of platelets affecting the glycoprotein Ib–von Willebrand factor axis. The von Willebrand factor multimer pattern in patients also appeared abnormal.
Conclusions: Multimodal antiplatelet monitoring showed markedly impaired platelet function in patients with a left ventricular assist device. Platelet dysfunction under high shear rates and abnormal ristocetin-induced aggregation is only partly attributable to low von Willebrand factor activity. These findings resemble the acquired von Willebrand syndrome that is associated with microaggregate formation and enhanced bleeding.
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Introduction |
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Foreign surfaces of LVADs, altered rheologic conditions, and blood stasis within the chambers of the native heart induce activation of coagulation [3] and require adequate anticoagulation to prevent thromboembolic events [4]. Most often, a combination of platelet inhibitors and a vitamin K antagonist is prescribed for this purpose [5]. Whereas clinicians routinely measure the effect of warfarin (Coumadin) derivatives on plasmatic coagulation, they rarely measure the effect of platelet inhibitors on platelet function. Severe perioperative bleeding and also spontaneous hemorrhage have been reported in LVAD patients that cannot completely be explained by the anticoagulation regimen alone. It has been suspected that platelet dysfunction does play a role here. We thus hypothesized that platelet dysfunction may be present in LVAD patients owing to high shear forces at the artificial surfaces. Shear stress, ie, stress, which is applied parallel or tangential to the face of a material, is sensed by ligand-bound extracellular domains of integrins and converted to functional cellular responses such as the activation of platelets. Hereby, high shear–induced platelet plug formation is dependent on a functioning von Willebrand factor–glycoprotein Ib (vWF–GpIb) axis.
To date, however, there is no routine monitoring procedure for platelet inhibition, and platelet function has not even been systematically evaluated in this patient population. Different platelet function tests currently on the market (ie, thrombelastography [TEG], rotation thromboelastometry, and the platelet function analyzer-100) have been used for this purpose to a variable extent by centers that have an active LVAD program. The three devices mentioned above use whole blood, which is a more physiologic approach as compared with studying platelet suspensions. The complementary use of these point-of-care tests allows assessment of the underlying pathophysiology of platelet dysfunction. However, with the exception of single case reports they have not been used simultaneously and test results have not been compared with each other. We therefore determined platelet function in LVAD outpatients with the help of the above mentioned and widely used four point-of-care tests including the newly developed Multiplate, a whole blood aggregometer. In addition, we also tried to elucidate the cause of the suspected dysfunction.
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Material and Methods |
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Thrombelastography Platelet Mapping Assay
Thrombelastography (Haemoscope, Skokie, IL) enables monitoring of hemostasis as a whole dynamic process, rather than isolated end points. Global assessment of hemostatic function is made from whole blood, documenting the interaction of platelets with the protein coagulation cascade from the time of the initial platelet–fibrin interaction, through platelet aggregation, clot strengthening, and fibrin cross linkage to eventual clot lysis. The "signature" of generated tracings can give information on clotting factor activity, platelet function, and any clinically significant fibrinolytic process within 20 to 30 minutes. The platelet-mapping assay that measures the presence of aspirin requires heparin to suppress thrombin and 10 µL of ActivatorF (Haemoscope) to replace thrombin in converting fibrinogen into fibrin and arachidonic acid as platelet agonist [6, 7]. ActivatorF consists of the snake venom reptilase that converts fibrinogen to des-A-fibrin and activated factor XIII, which crosslinks the fibrin monomers. Heparinized blood as well as ActivatorF is also necessary for the determination of fibrin to clot strength. In contrast, when kaolin is used as an activator, citrated blood is needed.
For quantification of TEG variables, the time from sample placement in the cuvette until the tracing amplitude reaches 2 mm represents the rate of initial fibrin formation and is related to plasma clotting factor and circulating inhibitor activity (intrinsic coagulation); the greatest amplitude on the TEG trace is a reflection of the absolute strength of the fibrin clot—a direct function of the maximum dynamic properties of fibrin and platelets.
Rotation Thromboelastometry
Rotation thromboelastometry (Pentapharm, Munich, Germany) is related to, but in some aspects different from, classical TEG. In rotation thromboelastometry a ball bearing guides the firmness sensor, which makes the analysis less susceptible to mechanical stress, movement, and vibration. Just before running the assay, citrated blood samples were recalcified with 20 µL of CaCl2 0.2 mol/L. To assess activation and inhibition of coagulation sensitively we did not add an exogenous agonist to the test system (no agonist thromboelastometry) [8]. The following variables were analyzed: coagulation time, clot formation time, and maximum clot firmness. To functionally assess the fibrinogen of the samples, the clotting was determined using tissue factor activation of clotting after addition of an unknown amount of platelet inhibitor cytochalasin D, which the company would not disclose (fibrinogen thromboelastometry). Both tests, no agonist and fibrinogen thromboelastometry, were performed in samples from both patients and volunteers.
The Platelet Function Analyzer-100
The platelet function analyzer-100 (Dade Behring, Marburg, Germany) measures in vitro platelet plug formation in whole blood under high shear stress. It determines the closure time needed for a platelet plug to form after activation of platelets by pathophysiologically relevant stimuli (eg, collagen adenosine diphosphate or collagen epinephrine). Aspirin prolongs the collagen epinephrine closure time [CEPI-CT] of activated blood, whereas the collagen adenosine diphosphate closure time [CADP-CT] is only minimally affected [9, 10].
The Multiplate Analyzer
The Multiplate analyzer (Dynabyte Medical, Munich, Germany) uses whole blood [10–12]. Test cells incorporate two independent sensor units, each consisting of two silver-coated 3.2-mm-long copper wires. The instrument detects the impedance change (aggregation) of each sensor separately. The most important variable is the area under the curve. Its unit is AU · min (as the y axis is the aggregation, expressed in aggregation units [AU], and the x axis is the time, expressed in minutes). One unit corresponds to 10 AU · min. As an anticoagulant, which does not affect the free calcium concentration in the sample, hirudin (a direct thrombin inhibitor) is preferred because heparin has some platelet-activating properties.
In the ristocetin test (triggered by 0.77 mg/mL ristocetin) the binding of ristocetin–vWF complexes leads to activation and aggregation of platelets, which relies on the binding of vWF to the GpIb receptor.
In the collagen test (triggered by 3.2 µg collagen/mL) platelets are activated by means of the collagen receptor. For appropriate platelet activation, release of endogenous arachidonic acid is necessary, which is then transformed to thromboxane A2 by cyclooxygenase. As cyclooxygenase is blocked by aspirin, the collagen test is sensitive to the action of aspirin. However, this mechanism also explains why the collagen test is less specific toward the action of aspirin compared with the aspirin test (ASPItest).
In the ASPItest, a defined amount of arachidonic acid is used as the activator, whereas in the collagen test arachidonic acid is formed by the platelet. Currently, only the ASPItest is recommended for monitoring aspirin responsiveness. Here, platelets are activated by arachidonic acid (0.5 mmol/L), which is converted by platelet cyclooxygenase to thromboxane A2. Aspirin can block cyclooxygenase, causing a reduced aggregation in the ASPItest.
In addition to these specific platelet function tests the following laboratory variables, which may directly or indirectly interact with platelet function, have been determined: coagulation factor VIII activity was assessed with a two-stage clotting method performed using the reagents from the two-stage factor VIII assay kit (Immuno, Vienna, Austria); total vWF antigen (vWF:Ag) was measured by an enzyme-linked immunosorbent assay (REAADS vWF:Ag Test Kit; Corgenix, Broomfield, CO); vWF ristocetin cofactor activity was assessed with an automated coagulometer (BCS-system; Dade-Behring, Marburg, Germany) using the vWF reagent (Dade-Behring) [13]; pro–brain natriuretic peptide was determined by an enzyme-linked immunosorbent assay kit (Immunodiagnostics, Bensheim, Germany); platelet counts were quantified with a SYSMEX cell counter (SYSMEX Counter 2100 XE; Milton Keynes, UK) from EDTA blood; and C-reactive protein was measured with the test "C-reactive protein Latex neu" (Olympus AU 5400; Olympus, Hamburg, Germany).
After having received the results from the above mentioned platelet function tests, we also determined vWF multimers in patients and control subjects to elucidate potential proteolysis or consumption of high molecular weight vWF multimers. Under high shear stress these multimers are most effective in platelet-mediated hemostasis. Gel electrophoresis with low-resolution gels (0.8%) was carried out using LGT agarose type VII (Sigma, Munich, Germany). The vWF multimers were transferred to nitrocellulose filters by electroblotting. Filters were incubated at room temperature in a 1:3000 dilution of polyclonal rabbit anti-human vWF–horseradish peroxidase antibody (Dako, Glostrup, Denmark). After several washing steps, SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was added to the filters. Detection of multimers was performed by using the luminescent image analyzer LAS 3000 (Fujifilm Life Science, Duesseldorf, Germany).
Sample Size Calculation and Statistical Analysis
No formal sample size calculation was performed, but we planned to enroll all eligible 12 outpatients who, at that time, were managed by the LVAD outpatient clinic. The only exclusion criterion was participation in another clinical trial. For reasons of robustness only nonparametric tests were applied. Comparison between groups was done with the Mann-Whitney U test. Correlations were calculated with the Spearman ranks correlation test. A probability value of less than 0.05 was considered significant for the exploratory nature of this investigation. Data are either presented as absolute values or means (± standard deviation).
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Platelet function as measured by the platelet function analyzer-100 was invariably and severely compromised: CADP-CT was 2.5-fold longer in patients (p < 0.001; Fig 1; Table 3), and 5 of 12 patients had maximal CADP-CT values of 300 seconds. This is in sharp contrast to the upper limit values of the CEPI-CT and CADP-CT ranges, which are 193 seconds and 160 seconds, respectively [14]. Even higher normal closure time values have been reported [9]. Levels of vWF:Ag were 80% higher in patients than in volunteers (p < 0.001; Table 2). In contrast, vWF ristocetin (vWF:RiCO) was subnormal (<60%) in only 5 of 12 patients (Fig 1; Table 2). Thus, low vWF activity can only account for a prolonged CADP-CT in these 5 patients, so that the usually good correlation between vWF:RiCO and CADP-CT, like in volunteers (r 2 = 0.90, p < 0.001), does not exist in LVAD patients (r 2 = 0.52; p = 0.084) [9]. By chance, there was also a high number of healthy subjects with low levels of vWF:RiCO. Ristocetin-induced aggregation was threefold higher in volunteers (p < 0.001; Fig 1; Table 3). Except for 1 patient there was no data overlap between patients and volunteers. Hence, ristocetin-induced aggregation was also abnormal in 6 of 7 patients with normal vWF:RiCO. Mean fluorescence intensity of GpIb on platelets as measured by flow cytometry was not reduced below control values (n = 8; data not shown). Typical immunoblots of the electrophoresis gels are depicted in Figure 2. These results indicate that the majority of the 12 LVAD patients appear to have an abnormal multimer pattern with depletion of high molecular weight multimers.
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Our LVAD patients who were treated with 100 mg of aspirin once a day had mean values in the ASPItest of 25 U. Calatzis and coworkers [15] regarded values less than 30 U as a preliminary cutoff for complete platelet inhibition by aspirin. Our own unpublished data of patients who took aspirin because of coronary artery disease showed a mean value of 27 U. Similar strong inhibition of arachidonic acid–induced platelet function was seen in the TEG assay (Table 3) in our LVAD patients. Thus there was no evidence for a diminished response to aspirin in LVAD patients.
Increased levels of fibrinogen, coagulation factor VIII activity, and vWF:Ag are associated with an acute-phase response and may indicate increased thrombotic risk [16]. In line with this and the observation that inflammation induces vWF release [17] is the correlation between vWF:Ag and plasma fibrinogen levels in our study. Furthermore, elevated vWF:Ag and activity are also established markers of cardiovascular risk [18].
In contrast to the greater than 60% enhanced vWF levels, the vWF:RiCO levels were subnormal in 5 of 12 LVAD patients (Fig 1). On the other hand, ristocetin-induced aggregation in the Multiplate was reduced in all but 1 patient (Fig 1). This discrepancy may be surprising because in both assays ristocetin causes platelet aggregation. However, there is a profound difference between the two assays. Ristocetin-cofactor activity is analyzed with the Multiplate by using the patients' own platelets, whereas the vWF:RiCO assay uses lyophilized, stabilized platelets from healthy donors. In the Multiplate analysis platelets have to stick to the impedance sensors to be detected and this requires amplification of aggregation by endogenous agonists, such as the formation of thromboxane A2. This is not necessary in the vWF:RiCO assay and may explain the inhibition observed in the Multiplate ristocetin test that was not reflected by vWF:RiCO levels in the aspirin-treated cohort.
The prolongation of CEPI-CT in the platelet function analyzer-100 analysis using collagen epinephrine is consistent with the aspirin therapy of patients. The prolongation of the CADP-CT (Fig 1), however, cannot be explained by aspirin as this test is rather insensitive toward aspirin. As adenosine diphosphate–induced aggregation was not impaired, a deficit in the adenosine diphosphate pathway could be ruled out. The prolonged CADP-CT in LVAD patients would rather indicate an acquired qualitative platelet function defect. This suggests that high shear–induced platelet plug formation is impaired, which depends on the vWF–GpIb axis. This is supported by the lack of the expected correlation between CADP-CT and vWF:RiCO [9], which is only seen in volunteers but not in patients. However, flow cytometric analysis of GpIb did not reveal decreased surface expression of GpIb as compared with volunteers (data not shown). In combination with reduced responsiveness in the Multiplate assay it is thus conceivable that the GpIb pathway is impaired.
The mechanical destruction of large vWF multimers may be of relevance in conditions in which shear rates are increased [19]. These blood flow conditions could also lead to binding of vWF to platelet GpIb receptors, causing formation of small platelet aggregates with subsequent consumption of large vWF multimers and platelets [20]. Depletion of high-molecular weight multimers in our LVAD patients would argue for either ongoing destruction or consumption of these large vWF multimers in LVAD outpatients even late after LVAD implantation. Mechanical trauma of red blood cells was supported by the characteristic increase in hemolysis markers. In addition, mechanical alteration of platelet function has previously been described as a consequence of extracorporeal circulation [21], aortic valve replacement [22], and, just recently, early after LVAD implantation [23]. Our observation of platelet dysfunction confirms and extends these findings of Geisen and colleagues [23]. Thus, mechanical trauma may decrease both vWF:RiCO and platelet function. Although there are certain minor technical differences between the three LVAD devices, the numbers in each group are too small to comment on the impact the specific material and the inner lining have on platelet function [24–26]. Therefore, no recommendations can be given so far regarding this issue. In addition, potential modifications of the antiplatelet regimen still have to be evaluated under the light of the current findings.
In summary, these data indicate that LVAD patients have elevated markers of inflammation, a markedly impaired platelet function under high shear rates, and impairment of ristocetin-induced aggregation, which are only partly attributable to a low vWF activity. Furthermore, disruption of high-molecular weight multimers in LVAD patients could be detected. This defect resembles the acquired von Willebrand syndrome, which can either cause platelet microaggregate formation or spontaneous and enhanced bleeding. In the case of augmented bleeding a potential benefit from the administration of desmopressin, which liberates intact vWF from platelets and endothelial cells or concentrates, should be evaluated. It could be a therapeutic option unless vWF stores have already been emptied. The use of desmopressin at the dosage recommended for the treatment of von Willebrand disease (ie, 0.3 µg/kg) is associated with little side effects and might be a potential remedy during conditions of increased bleeding. An alternative approach could be the use of vWF concentrates in instances in which high molecular weight vWF multimers are perpetually and extensively disrupted by high shear forces.
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