Clinical Hemorheology and Microcirculation 61 (2015) 417–427 DOI 10.3233/CH-141866 IOS Press
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Plasma viscosity, functional fibrinogen, and platelet reactivity in vascular surgery patients Marco Ranucci∗ , Matteo Ranucci, Tommaso Laddomada, Ekaterina Baryshnikova, Giovanni Nano and Santi Trimarchi Department of Cardiothoracic – Vascular Anesthesia and Intensive Care, Department of Vascular Surgery, IRCCS Policlinico San Donato, Milan, Italy
Abstract. BACKGROUND: Platelet reactivity changes with shear stress, which in turn depends on whole blood and plasma viscosity (PV). Platelets interact with fibrinogen during thrombus formation, and fibrinogen is a determinant of PV. The respective role of PV and fibrinogen on platelet function is still unclear. METHODS: 30 patients undergoing vascular surgery were admitted to this study. In each patient we measured PV using a coneon-plate viscosimeter, functional fibrinogen using thromboelastometry, and platelet reactivity to thrombin receptor activating peptide (TRAP) stimulation using multi-electrode aggregometry. Routine coagulation parameter were measured. RESULTS: At the univariate analysis, platelet reactivity was positively associated with mean platelet volume (R2 = 0.15, P = 0.033) and PV (R2 = 0.35, P = 0.0006), and negatively associated with serum bilirubin (R2 = 0.20, P = 0.013) and international normalized ratio (INR) (R2 = 0.19, P = 0.017). At the multivariable analysis, only PV (P = 0.001) and INR (P = 0.019) remained independent predictors of platelet reactivity. CONCLUSION: PV is directly and independently associated with platelet reactivity, whereas functional fibrinogen is not. Aspirin treatment is inadequate to correct thrombin-induced platelet aggregation. In presence of hyperviscosity, patients at high cardiovascular risk, may benefit from more aggressive anti-platelet treatments. Keywords: Plasma viscosity, fibrinogen, platelet reactivity, thrombin
1. Introduction Plasma viscosity (PV) is a recognized factor contributing to platelet activation. The mechanism linking PV to platelet aggregation is the increased shear force at the vessel wall induced by high levels of PV [16, 19, 21, 28]. A high mean platelet volume (MPV), suggestive for increased platelet reactivity [1, 4], is associated with increased levels of PV [23]. Fibrinogen is an important determinant of PV [15], but high levels of fibrinogen have been associated with platelet activation as well [2, 10]. All together, PV [13, 18], MPV [14, 30], platelet reactivity [17, 32], and fibrinogen levels [29, 31], are recognized risk factors for cardiovascular risk, including ischemic heart disease and stroke. Given the association between fibrinogen levels and PV, and the ability of fibrinogen to improve platelet aggregation, it is presently unclear the respective role of PV and fibrinogen in determining ∗
Corresponding author: Marco Ranucci, MD, FESC, Director of Clinical Research in the Department of Anesthesia and Intensive Care, IRCCS Policlinico San Donato, Via Morandi 30, 20097 San Donato Milanese, Milan, Italy. Tel.: +39 02 52774320; Fax: +39 02 55602262; E-mail: [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
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platelet reactivity. Additionally, studies addressing the relationship between PV and platelet activation have been using the MPV as an indirect marker of platelet activation, but studies directly assessing platelet reactivity at different levels of PV are still lacking. Therefore, we have designed a prospective cohort study focused on vascular surgery patients, with the aim to determine the relationship of PV and fibrinogen with platelet reactivity, directly measured with a point-of-care aggregometer. 2. Materials and methods This study was approved by the local ethics committee (IRCCS San Raffaele Hospital), as a part of a general study addressing whole blood and plasma viscosity in surgical patients. All the patients provided a written informed consent. 2.1. Patients and sample size Thirty patients undergoing vascular surgery operations were admitted to the study. The general characteristics of the patient population are listed in Table 1. Exclusion criteria were the use of antiplatelet agents inhibiting the P2 Y12 receptor (ticlopidine, clopidogrel, prasugrel, ticagrelor) or the GPIIbIIIa receptor (abciximab, eptifibatide, tirofiban). Use of aspirin and/or low molecular weight heparin was allowed. Other exclusion criteria were the use of unfractionated heparin, warfarin, direct thrombin inhibitors, the presence of known congenital coagulation defects and an age below 18 years. Sample size was assessed by a preliminary power analysis, based on a regression model having one independent variable (PV), an anticipated effect size of 0.30, a statistical power of 0.80, and a probability level of 0.05. These data provided a minimum number of 28 subjects; taking into account the possibility of missing data, the sample size was settled at 30 subjects. 2.2. Procedures For every patient, the standard coagulation tests were available from the preoperative standard laboratory examinations. These included prothrombin time with international normalized ratio (INR), activated partial thromboplastin time (aPTT), platelet count and MPV. Immediately before surgery, an amount of 3 mL of blood was withdrawn from a venous or arterial line and collected into tubes containing hirudin, a direct thrombin inhibitor anticoagulant. The blood was used to measure platelet aggregation and fibrinogen concentration at thromboelastometry (ROTEM) using the specific FIBTEM test. The remaining blood was centrifugated at 3,200 rpm for 10 minutes, obtaining platelet poor plasma. An amount of 0.5 mL of platelet poor plasma was used to assess PV. 2.3. Measurements Platelet reactivity was tested using a multi-electrode aggregometry (Multiplate® , Verum Diagnostica GmbH, M¨unchen, Germany). An amount of 300 l blood was added to 300 l 37◦ C preheated saline solution and platelet aggregation was analyzed after activation with the commercially available reagent thrombin receptor-activating peptide (TRAP-6, TRAPtest, 32 M final concentration). TRAP-6 is a potent platelet activator via the thrombin receptor, which is normally not inhibited by intake of aspirin
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Table 1 General details of the patient population (N = 30) Variable Gender Male Female Age (years) Weight (kgs) Serum albumin (g/dL) Serum bilirubin (mg/dL) Serum creatinine (mg/dL) Diabetes Hypertension Dyslipidemia Smoking habit Chronic obstructive pulmonary disease Obstructive sleep apnea syndrome Active bloodstream infection Atrial fibrillation Previous vascular surgery Drug therapy Aspirin Low-molecular weight heparin treatment Statins Calcium channels blockers Beta-blockers Angiotensin converting enzyme inhibitors Diuretics Type of surgery Carotid artery thromboendoarterectomy Endovascular aneurysm repair Open aneurysm repair Peripheral arterial surgery
Number (%) or mean (standard deviation) 22 (73.3) 8 (26.7) 65 (21) 71 (12.3) 4.3 (0.4) 0.56 (0.3) 1.1 (1.1) 10 (33.3) 23 (76.7) 18 (60) 13 (43.3) 5 (16.7) 1 (3.3) 1 (3.3) 3 (10) 10 (33.3) 23 (76.7) 10 (33.3) 15 (50) 12 (40) 20 (66.6) 18 (60) 13 (43.3) 11 (36.6) 7 (23.3) 4 (13.3) 8 (26.7)
and only partially inhibited by thienopyridines or other related drugs. Conversely, TRAPtest is sensitive to GPIIbIIIa inhibitors, and in absence of these drugs represents the “natural” aggregation potential of the platelets. Increasing electric impedance was electronically measured for 6 minutes and expressed as the area under the aggregation curve plotted over time (AUC, [U]) by an integrated software. Reference ranges indicated by the manufacturer were AUCs 94 [U] to 156 [U] for TRAPtest. Functional fibrinogen was assessed using the FIBTEM test at the ROTEM delta (Tem International GmbH, Munich, Germany). Using the automated ROTEM pipette, 20 L of FIBTEM reagent were added to the cup, followed by 300 L of blood. The test was started automatically via the pipette signal. The reaction mixture (a total of 340 L) was withdrawn and then pipetted back into the cup, and the cup was set onto the pin. The maximum clot firmness (MCF) in millimeters was measured and considered a
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value for functional fibrinogen. With respect to fibrinogen concentration measured with the usual Clauss method, this approach is based on whole-blood viscoelastic coagulation assessment, which demonstrates the contribution to clot strength not only of fibrinogen but also elements such as red blood cells and factor XIII. However, many studies have demonstrated a significant correlation between FIBTEM values and Clauss fibrinogen concentration [8, 11, 20, 22]. Plasma viscosity was measured using a cone-on-plate Brookfield DV3T viscosimeter (Brookfield Engineering Laboratories, Stoughton, MA, USA). 500 L of plasma were placed on the surface of the plate using a calibrated micropipette; the surface of the cone-on-plate system was maintained at a temperature of 37◦ C using a heat-exchanger Heater Unit HU 35 Maquet (Getinge Group, Getinge, Sweden). The spindle of the cone-on-plate system was equipped with a cone model CPA 40Z. Plasma viscosity was assessed after a stabilization time of 2 minutes, at a rotation speed of 100 rpm, corresponding to a shear rate of 750 S−1 , and expressed in centipoise (cP). The conventional coagulation tests were performed by the hospital laboratory, and included platelet count, MPV, aPTT and INR. 2.4. Statistical analysis All data are expressed as mean and standard deviation for continuous, normally distributed variables; median and interquartile range for continuous, non-normally distributed variables; and number and percentage for categorical variables. Normality of the distribution was checked with a Kolgomorov-Smirnov test. The potential determinants of platelet reactivity were investigated for association with platelet reactivity using regression analyses for continuous variables, where different equations were tested and the function with the highest level of association was chosen to explain the phenomenon. Correlation coefficients and P-values were used to assess the best-fit among the various functions explored. For binary variables, the association with platelet reactivity was explored using a Student’s t-test. In case of multiple variables being associated with platelet reactivity, multivariable analyses (stepwise forward regression analysis) were applied, including all the variables being associated with platelet reactivity at a P value
417
Plasma viscosity, functional fibrinogen, and platelet reactivity in vascular surgery patients Marco Ranucci∗ , Matteo Ranucci, Tommaso Laddomada, Ekaterina Baryshnikova, Giovanni Nano and Santi Trimarchi Department of Cardiothoracic – Vascular Anesthesia and Intensive Care, Department of Vascular Surgery, IRCCS Policlinico San Donato, Milan, Italy
Abstract. BACKGROUND: Platelet reactivity changes with shear stress, which in turn depends on whole blood and plasma viscosity (PV). Platelets interact with fibrinogen during thrombus formation, and fibrinogen is a determinant of PV. The respective role of PV and fibrinogen on platelet function is still unclear. METHODS: 30 patients undergoing vascular surgery were admitted to this study. In each patient we measured PV using a coneon-plate viscosimeter, functional fibrinogen using thromboelastometry, and platelet reactivity to thrombin receptor activating peptide (TRAP) stimulation using multi-electrode aggregometry. Routine coagulation parameter were measured. RESULTS: At the univariate analysis, platelet reactivity was positively associated with mean platelet volume (R2 = 0.15, P = 0.033) and PV (R2 = 0.35, P = 0.0006), and negatively associated with serum bilirubin (R2 = 0.20, P = 0.013) and international normalized ratio (INR) (R2 = 0.19, P = 0.017). At the multivariable analysis, only PV (P = 0.001) and INR (P = 0.019) remained independent predictors of platelet reactivity. CONCLUSION: PV is directly and independently associated with platelet reactivity, whereas functional fibrinogen is not. Aspirin treatment is inadequate to correct thrombin-induced platelet aggregation. In presence of hyperviscosity, patients at high cardiovascular risk, may benefit from more aggressive anti-platelet treatments. Keywords: Plasma viscosity, fibrinogen, platelet reactivity, thrombin
1. Introduction Plasma viscosity (PV) is a recognized factor contributing to platelet activation. The mechanism linking PV to platelet aggregation is the increased shear force at the vessel wall induced by high levels of PV [16, 19, 21, 28]. A high mean platelet volume (MPV), suggestive for increased platelet reactivity [1, 4], is associated with increased levels of PV [23]. Fibrinogen is an important determinant of PV [15], but high levels of fibrinogen have been associated with platelet activation as well [2, 10]. All together, PV [13, 18], MPV [14, 30], platelet reactivity [17, 32], and fibrinogen levels [29, 31], are recognized risk factors for cardiovascular risk, including ischemic heart disease and stroke. Given the association between fibrinogen levels and PV, and the ability of fibrinogen to improve platelet aggregation, it is presently unclear the respective role of PV and fibrinogen in determining ∗
Corresponding author: Marco Ranucci, MD, FESC, Director of Clinical Research in the Department of Anesthesia and Intensive Care, IRCCS Policlinico San Donato, Via Morandi 30, 20097 San Donato Milanese, Milan, Italy. Tel.: +39 02 52774320; Fax: +39 02 55602262; E-mail: [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
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M. Ranucci et al. / Plasma viscosity and platelet reactivity
platelet reactivity. Additionally, studies addressing the relationship between PV and platelet activation have been using the MPV as an indirect marker of platelet activation, but studies directly assessing platelet reactivity at different levels of PV are still lacking. Therefore, we have designed a prospective cohort study focused on vascular surgery patients, with the aim to determine the relationship of PV and fibrinogen with platelet reactivity, directly measured with a point-of-care aggregometer. 2. Materials and methods This study was approved by the local ethics committee (IRCCS San Raffaele Hospital), as a part of a general study addressing whole blood and plasma viscosity in surgical patients. All the patients provided a written informed consent. 2.1. Patients and sample size Thirty patients undergoing vascular surgery operations were admitted to the study. The general characteristics of the patient population are listed in Table 1. Exclusion criteria were the use of antiplatelet agents inhibiting the P2 Y12 receptor (ticlopidine, clopidogrel, prasugrel, ticagrelor) or the GPIIbIIIa receptor (abciximab, eptifibatide, tirofiban). Use of aspirin and/or low molecular weight heparin was allowed. Other exclusion criteria were the use of unfractionated heparin, warfarin, direct thrombin inhibitors, the presence of known congenital coagulation defects and an age below 18 years. Sample size was assessed by a preliminary power analysis, based on a regression model having one independent variable (PV), an anticipated effect size of 0.30, a statistical power of 0.80, and a probability level of 0.05. These data provided a minimum number of 28 subjects; taking into account the possibility of missing data, the sample size was settled at 30 subjects. 2.2. Procedures For every patient, the standard coagulation tests were available from the preoperative standard laboratory examinations. These included prothrombin time with international normalized ratio (INR), activated partial thromboplastin time (aPTT), platelet count and MPV. Immediately before surgery, an amount of 3 mL of blood was withdrawn from a venous or arterial line and collected into tubes containing hirudin, a direct thrombin inhibitor anticoagulant. The blood was used to measure platelet aggregation and fibrinogen concentration at thromboelastometry (ROTEM) using the specific FIBTEM test. The remaining blood was centrifugated at 3,200 rpm for 10 minutes, obtaining platelet poor plasma. An amount of 0.5 mL of platelet poor plasma was used to assess PV. 2.3. Measurements Platelet reactivity was tested using a multi-electrode aggregometry (Multiplate® , Verum Diagnostica GmbH, M¨unchen, Germany). An amount of 300 l blood was added to 300 l 37◦ C preheated saline solution and platelet aggregation was analyzed after activation with the commercially available reagent thrombin receptor-activating peptide (TRAP-6, TRAPtest, 32 M final concentration). TRAP-6 is a potent platelet activator via the thrombin receptor, which is normally not inhibited by intake of aspirin
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Table 1 General details of the patient population (N = 30) Variable Gender Male Female Age (years) Weight (kgs) Serum albumin (g/dL) Serum bilirubin (mg/dL) Serum creatinine (mg/dL) Diabetes Hypertension Dyslipidemia Smoking habit Chronic obstructive pulmonary disease Obstructive sleep apnea syndrome Active bloodstream infection Atrial fibrillation Previous vascular surgery Drug therapy Aspirin Low-molecular weight heparin treatment Statins Calcium channels blockers Beta-blockers Angiotensin converting enzyme inhibitors Diuretics Type of surgery Carotid artery thromboendoarterectomy Endovascular aneurysm repair Open aneurysm repair Peripheral arterial surgery
Number (%) or mean (standard deviation) 22 (73.3) 8 (26.7) 65 (21) 71 (12.3) 4.3 (0.4) 0.56 (0.3) 1.1 (1.1) 10 (33.3) 23 (76.7) 18 (60) 13 (43.3) 5 (16.7) 1 (3.3) 1 (3.3) 3 (10) 10 (33.3) 23 (76.7) 10 (33.3) 15 (50) 12 (40) 20 (66.6) 18 (60) 13 (43.3) 11 (36.6) 7 (23.3) 4 (13.3) 8 (26.7)
and only partially inhibited by thienopyridines or other related drugs. Conversely, TRAPtest is sensitive to GPIIbIIIa inhibitors, and in absence of these drugs represents the “natural” aggregation potential of the platelets. Increasing electric impedance was electronically measured for 6 minutes and expressed as the area under the aggregation curve plotted over time (AUC, [U]) by an integrated software. Reference ranges indicated by the manufacturer were AUCs 94 [U] to 156 [U] for TRAPtest. Functional fibrinogen was assessed using the FIBTEM test at the ROTEM delta (Tem International GmbH, Munich, Germany). Using the automated ROTEM pipette, 20 L of FIBTEM reagent were added to the cup, followed by 300 L of blood. The test was started automatically via the pipette signal. The reaction mixture (a total of 340 L) was withdrawn and then pipetted back into the cup, and the cup was set onto the pin. The maximum clot firmness (MCF) in millimeters was measured and considered a
420
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value for functional fibrinogen. With respect to fibrinogen concentration measured with the usual Clauss method, this approach is based on whole-blood viscoelastic coagulation assessment, which demonstrates the contribution to clot strength not only of fibrinogen but also elements such as red blood cells and factor XIII. However, many studies have demonstrated a significant correlation between FIBTEM values and Clauss fibrinogen concentration [8, 11, 20, 22]. Plasma viscosity was measured using a cone-on-plate Brookfield DV3T viscosimeter (Brookfield Engineering Laboratories, Stoughton, MA, USA). 500 L of plasma were placed on the surface of the plate using a calibrated micropipette; the surface of the cone-on-plate system was maintained at a temperature of 37◦ C using a heat-exchanger Heater Unit HU 35 Maquet (Getinge Group, Getinge, Sweden). The spindle of the cone-on-plate system was equipped with a cone model CPA 40Z. Plasma viscosity was assessed after a stabilization time of 2 minutes, at a rotation speed of 100 rpm, corresponding to a shear rate of 750 S−1 , and expressed in centipoise (cP). The conventional coagulation tests were performed by the hospital laboratory, and included platelet count, MPV, aPTT and INR. 2.4. Statistical analysis All data are expressed as mean and standard deviation for continuous, normally distributed variables; median and interquartile range for continuous, non-normally distributed variables; and number and percentage for categorical variables. Normality of the distribution was checked with a Kolgomorov-Smirnov test. The potential determinants of platelet reactivity were investigated for association with platelet reactivity using regression analyses for continuous variables, where different equations were tested and the function with the highest level of association was chosen to explain the phenomenon. Correlation coefficients and P-values were used to assess the best-fit among the various functions explored. For binary variables, the association with platelet reactivity was explored using a Student’s t-test. In case of multiple variables being associated with platelet reactivity, multivariable analyses (stepwise forward regression analysis) were applied, including all the variables being associated with platelet reactivity at a P value
