TR-05503; No of Pages 7 Thrombosis Research xxx (2014) xxx–xxx
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Regular Article
Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheresis Tomasz Rusak a, Jarosław Piszcz b, Tomasz Misztal a, Justyna Brańska-Januszewska c, Marian Tomasiak a,⁎ a b c
Department of Physical Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland Department of Hematology, Medical University of Bialystok, Skłodowskiej-Curie 24A, 15-276 Bialystok, Poland Department of Biology, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland
a r t i c l e
i n f o
Article history: Received 11 March 2014 Received in revised form 4 April 2014 Accepted 23 April 2014 Available online xxxx Keywords: Clot retraction Clot structure Fibrinolysis Platelets Polycythemia vera Thromboelastometry
a b s t r a c t Using patients with polycythemia vera (PV) as an experimental model, we evaluated the impact of clot retraction (CR) and architecture of the clot on fibrinolysis. We studied the kinetics of clot retraction and the fibrinolysis rate in whole blood from 48 PV patients and 48 healthy controls. Measurements were performed before and after isovolemic eryhrocytapheresis (ECP). CR was measured by optical method. Clot lysis time (CLT) and maximum clot firmness (MCF) were measured by thromboelastometry in recalcified blood supplemented with t-PA and tissue factor. Compared with healthy controls, CR rate in PV patients was higher (0.0219 vs. 0.0138; p b 0.001), the clot volume smaller and MCF elevated (64 vs. 58 mm). CR rate correlated with platelet count (r = 0.546; p = 0.001) but not with erythrocyte concentration (r = 0.192; p N 0.3). Compared with healthy controls, CLT in PV patients was significantly prolonged (158 min vs. 71 min). Fibrinolysis rate inversely correlated with CR rate (r = -0.566; p b 0.001); MCF (r = -0.704; p b 0.001) and platelet count (r = -0.461; p b 0.001). As judged by confocal microscope, in comparison to healthy controls, clots formed in blood from PV patients demonstrated booth a distinctly higher degree of crosslinking and possessed thinner fibers. Altered CR, MCF and fibrinolysis speeds were not normalized following the ECP procedure. Tirofiban (a blocker of platelet GPIIb/IIIa receptors), unlike aspirin, normalized abnormal CR and fibrinolysis in blood from PV patients. Collectively, our data indicate that in PV patients, abnormal CR may result in formation of thrombi that are more resistant to fibrinolysis. ECP and aspirin failed to normalize platelet-related fibrinolysis resistance. © 2014 Elsevier Ltd. All rights reserved.
Introduction Clot retraction is defined as the slow shrinking of a freshly formed platelet-fibrin clot closely attached to the injured blood vessel wall [1–4]. Retraction of the clot makes possible faster recanalization of the occluded (by thrombus) blood vessel which may result in shortening the time of ischemia of neighboring tissues. The retracted clot is more strongly connected with the vessel wall, more stable mechanically, and thus less prone to detaching under conditions of high shear stress [2]. Clot retraction is also proposed to have great impact on the lysis of a freshly formed platelet-fibrin clot. Studies, carried out on models of fibrinolysis and thrombolysis that properly mimic physiological Abbreviations: ASA, aspirin; CR, clot retraction; CLT, clot lysis time; ECP, erythracytapheresis procedure; MCF, maximum clot firmness; PAI-1, plasminogen activator inhibitor-1; PV, polycythemia vera; PRP, platelet rich plasma; RBC, red blood cells; TF, tissue factor; tPA, tissue plasminogen activator; WBC, white blood cells. ⁎ Corresponding author. Tel.: +48 85 748 5714; fax: +48 85 748 5416. E-mail addresses: [email protected] (T. Rusak), [email protected] (J. Piszcz), [email protected] (T. Misztal), [email protected] (J. Brańska-Januszewska), [email protected] (M. Tomasiak).
conditions [2,5–7], strongly indicate that platelets make clots resistant to lysis. This phenomenon has been termed “platelet-mediated fibrinolysis resistance”. The mechanism(s) by which human platelets make clots resistant to fibrinolysis are not completely understood, but the accumulated evidence points to clot retraction as one of the major causes. Understanding the cross-talk between clot retraction and fibrinolysis is of great clinical importance since reactive nitrogen species released by activated inflammatory cells have recently been reported to modulate clot retraction rate [8,9]. Although the link between clot retraction and fibrinolysis velocity in vitro is well documented [10–12], much less is known about a similar relationship in clinical conditions characterized with altered hemostasis. A prominent example of such a condition is polycythemia vera. Polycythemia vera (PV) is a chronic myeloproliferative neoplasm, characterized by increased red cell mass and is often accompanied by thrombocytosis and leukocytosis [13–15]. The clinical course of the disease can be complicated by both thrombotic and hemorrhagic events which remain the leading causes of morbidity and mortality in untreated PV patients [15,16]. What’s more, both altered clot retraction and fibrinolysis have been reported in PV patients [17,18].
http://dx.doi.org/10.1016/j.thromres.2014.04.025 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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Consequently, this study was undertaken to evaluate the link between clot retraction and fibrinolysis in blood from patients suffering from PV. Another aim was to determine whether normalization of the erythrocyte count by means of routinely performed isovolemic erythrocytapheresis (ECP) affects clot retraction and fibrinolysis in patients suffering from PV. Materials and methods Study subjects Forty eight patients (18 men, 30 women) with PV, diagnosed according to criteria established by the World Health Organization [19], and an age-matched control group of 48 healthy volunteers (19 men, 29 women) with normal blood cell counts were entered into the study. Thirty six PV patients took low dose aspirin, whereas 12 of them discontinued aspirin therapy at least one week before blood collection. At the moment of blood collection, patients enrolled in the study did not undergo pharmacological cytoreductive therapy. Healthy volunteers had not taken medication known to affect platelet function and/or coagulation for at least 10 days before blood sampling. None of the study subjects had taken oral contraceptives or hormone replacement therapy for at least two months before blood collection. The study protocol was approved by the Ethics Committee at the Medical University of Bialystok. The procedures were in accordance with the Declaration of Helsinki of 1975, as revised in 2000 and blood samples were obtained with the subjects’ informed consent. Blood collection and erythrocytapheresis procedure Venous blood was collected with minimum trauma and stasis via a 21-gauge needle (0.8 × 40 mm) into 9 ml polypropylene vacuum tubes (Vacuette, Greiner Bio-One, Kremsmünster, Austria) containing 130 mM trisodium citrate. All PV patients were undergoing erythrocytapheresis (ECP), which was carried out using MCS + cell separator (Haemonetics, Braintree, MA, USA). The removed erythrocyte volume (100-410 ml) was replaced with an equal amount of physiological saline as fluid compensation. The instrument has software which, based on the initial hematocrit, body weight and sex of the subject, precisely determines the blood volume and hematocrit at the end of the procedure. Blood was collected 20 minutes before ECP and an hour after the procedure.
Evaluation of platelet-fibrin clot lysis by fluorimetric method Lysis of platelet-fibrin clots was estimated according to Boulaftali et al. with some modifications [20]. PRP samples (0.4 ml) were supplemented with Alexa Fluor 488-labeled human fibrinogen (final conc. 75 μg/ml) and incubated at 37 °C for 2 minutes. Afterwards, aliquots of 0.1 ml were transferred to plastic tubes containing 0.775 ml of Tyrode-Hepes buffer supplemented with CaCl2 (final conc. 10 mM) and tissue factor (Innovin, final conc. as indicated), stirred for 30 sec and incubated at 37 °C for 1 hour in the dark to allow full clot retraction. To perform lysis, the retracted clots were transferred to polypropylene tubes containing a 1.5 ml of fresh T-H buffer and incubated at 37 °C for 24 hours in the dark. Then, samples were centrifuged (5 min, 11000 ×g, room temperature) to remove clots, and the aliquots (500 μl) of supernatant were collected for determination of fluorescence intensity (Ex 488 nm, Em 522 nm) associated with Alexa Fluor 488containing fibrin degradation products (AF488-FDP), using the Hitachi F-7000 fluorimeter (Hitachi Ltd., Japan). Fluorescence intensity reflects the progress of fibrinolysis. To determine the maximal fibrinolysis (100% lysis) the retracted clots were incubated 37 °C for 24 hours in the dark with tPA added to the final concentration of 1 μg/ml. In some experiments, PRP samples were incubated with aspirin (final conc. 500 μM) or tirofiban (final conc. 200 μg/ml) for 10 min before the addition of Alexa Fluor 488-fibrinogen. Confocal microscopy of platelet-fibrin clots Samples (100 μl) of PRP were supplemented with Alexa Fluor 488labeled human fibrinogen (0.15 μM final conc., approximately 15 dye molecules for each fibrinogen molecule) and preincubated for 2 min. at 37 °C. Next, CaCl2 (20 mM final conc.) or CaCl2 + recombinant tissue factor (10 mM and 140 ng/ml final conc. respectively) were added, mixed vigorously and aliquots of 25 μL were transferred to microchamber slides (Ibidi μ-slide VI; Thistle Scientific). Clotting samples were incubated for 2 h at 37 °C in humid atmosphere, protected from light. After gelation time, clots architecture was imaged using fluorescence microscope with confocal imaging system – Nikon ECLIPSE Ti/C1 Plus (Ex 488 nm/Em 515/30 nm, 100× magnification). The images were acquired with a field of view of 120 × 120 μm. At least 10 pictures of different areas of each clot was taken and one representative image is presented.
Measurement of kinetics of clot retraction Routine hematological assays Measurement of the kinetics of clot retraction in whole blood were performed in non-siliconized glass tubes as described before [8]. Pictures were taken for one hour at 10 min intervals and after 120 min using a digital camera. Quantification of retraction was performed by assessment of the clot area by use of Motic Images Plus 2.0 ML software, and data were processed using Microsoft Excel. Clot surface areas were plotted as a percentage of maximal retraction (i.e. volume of platelet suspension). Data were expressed as follows: percentage of retraction (relative clot volume) = (area t0 − area t)/(area t0) × 100. Kinetics of clot retraction was characterized by the calculation of rate constant of retraction process. Thromboelastometric analyses Thromboelastometric measurements were performed on the ROTEM® system (Tem International GmbH, Manheim, Germany). Recalcified (10 mM CaCl2) blood was assessed for fibrinolytic potential using either 140 ng/ml tissue factor (TF), and 125 ng/ml of tissue plasminogen activator (tPA). We measured the parameters characterized clot strength (maximal clot firmness) and fibrinolysis (percentage reduction of MCF in time, clot lysis time). The ROTEM analysis was started 30 min after the blood was drawn by adding 320 μl of blood to a cuvette already containing 20 μl of re-calcification reagents.
Complete blood cell count, Hct and hemoglobin levels, were measured by an automated hematology analyzer (Sysmex SE9000, Toa Medical Electronics, Kobe, Japan). Data analysis Data reported in this paper are the median of the number of determinations indicated (n). Statistical analysis was performed by Mann-Whitney-Wilcoxon U test and elaboration of experimental data by the use of STATISTICA software (StatSoft, Tulsa, OK, USA). Differences were considered significant at a p value b 0.05. Correlations were assessed by a non-parametric test (Spearman’s rank correlation coefficient, r). Results Erythrocytapheresis-evoked changes in blood morphology The results presented in Table 1 show that the erythrocyte (RBC), leukocyte (WBC) and platelet counts of patients with PV were significantly greater than those of the control subjects. Erythrocytapheresis (ECP) resulted in a significant reduction in erythrocyte count, hematocrit and
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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Table 1 Characteristics of study subjects. Polycythemia vera (n = 48)
men/woman Age, median (range) RBC (×1012/l) WBC (×109/l) Platelets (×109/l) Haematocrit Hemoglobin (g/dl) Thromboelastometry: G (dynes/cm2) MCF (mm) LI30 (%): LI60 (%): LI90 (%): CLT (min):
Controls (n = 48)
before ECP
after ECP
18/30 58.9 (43 ÷ 75) 6.04 (4.51 ÷ 9.07) ⁎⁎ 10.0 (6.0 ÷ 23.3) ⁎⁎ 394 (183 ÷ 792)⁎ 0.533 (.465 ÷ .627) ⁎⁎ 17.3 (14.6 ÷ 22.4)⁎⁎
5.36 (4.07 ÷ 7.21) ⁎, † 9.1 (4.6 ÷ 21.1)⁎ ⁎ 385 (182 ÷ 755)⁎ 0.467 (.402 ÷ .545)⁎, † 15.2 (12.4 ÷ 19.4) ⁎,†
19/29 57.2 (42 ÷ 71) 4.48 (4.03 ÷ 5.84) 6.3 (3.6 ÷ 10.9) 219 (115 ÷ 351) 0.412 (.358 ÷ .507) 13.7 (12.2 ÷ 17.0)
8973 (4636 ÷ 16408)⁎ 64 (51 ÷ 76)⁎ 0 (0 ÷ 5) ⁎ 11 (2 ÷ 49) ⁎⁎ 29 (6 ÷ 93) ⁎⁎ 158 (84 ÷ 268) ⁎
9426 (4898 ÷ 16376) ⁎ 65 (51 ÷ 74) ⁎ 0 (0 ÷ 3) ⁎ 10 (2 ÷ 55) ⁎⁎ 26 (8 ÷ 94) ⁎⁎ 156 (93 ÷ 242) ⁎
7001(3043 ÷ 10454) 58 (39 ÷ 68) 6 (1 ÷ 49) 93 (29 ÷ 100) 100 (58 ÷ 100) 71 (49 ÷ 116)
⁎) p b 0.05; ⁎⁎) p b 0.01 (compared with healthy subjects); ) p b 0.05 (compared with PV patients before ECP). G – the absolute shear elastic modulus of the sample is then calculated from the maximal clot firmness (MCF) as follows: G = 5000*MCF/(100-MCF). LI (Lysis Index) - a reduction of amplitude relative to MCF at 30, 60 and 90 min after the initiation of blood clotting. CLT – clot lysis time (time of complete fibrinolysis). †
hemoglobin values. The procedure practically was without any effect on platelet count which remained significantly higher than those in healthy controls.
Kinetics of clot retraction, effect of erythrocytapheresis Fig. 1 shows the results of experiments in which we compared the kinetics of CR (panel A) and the volume of retracted clots (panel B) in the blood of both PV patients and healthy control. As is seen, the rate constant of clot retraction was significantly higher in PV patients than in healthy controls (0.0219 vs. 0.0138 min-1; p b 0.001). Correspondingly compared with healthy subjects, PV patients have strongly reduced volume of retracted clots measured 40 minutes after initiation of clotting (0.44 vs. 0.56). ECP procedure failed to normalize the kinetics of clot retraction (k = 0.0217 min-1) and the volume of retracted clots (0.45) in PV patients. Fibrinolysis resistance evaluated by ROTEM rotational tromboelastometry Experiments shown in Table 1 were performed to evaluate the lysis speed of clots formed in blood from PV patients and healthy controls. The fibrinolytic response by tPA was assessed with the use of ROTEM software, thereby providing the lysis progress at 30, 60, and 90 minutes, complete lysis time, MCF and G variables. As is shown, compared to healthy controls, polycythemic patients have prolonged lysis times (158 vs. 71 min; p b 0.001) and augmented MCF (64 vs. 58 mm) and G (8973 vs. 7001 dynes/cm2; p b 0.001) variables, indicating the existence of a fibrinolysis resistance state. Neither MCF variable (65 mm) nor clot lysis time (156 min) were markedly changed following the erythrocytapheresis procedure, indicating its inefficacy in reducing fibrinolysis resistance. Architecture of platelet fibrin clots The results presented in Fig. 2 show the architecture of plateletfibrin clots formed in blood from healthy controls (panel A, C) and from PV patients (panel B, D) visualized with confocal microscope. As is seen, the fibrin network architecture of clots formed in blood from PV and from healthy controls dramatically differs. More specifically, compared with healthy subjects, PV patients have clots that are much more dense, more crosslinked, and are made from thinner fibers.
Fig. 1. Clot retraction in healthy subjects (control; n = 48) and PV patients (n = 48; before and after ECP procedure). Clot surface areas were assessed by digital processing and plotted as percentage of volume of blood suspension. Panel A shows volumes of clots measured 40 minutes after initiation of clotting. Panel B represents the clot retraction rates (to each patient) calculated from the slope of the clot volume against time (min-1).
Impact of thrombin formation rate on clot retraction and fibrinolysis speed To evaluate whether a pattern of thrombin generation affects clot retraction and fibrinolysis speed, in our experimental model we tested the impact of increasing concentrations of exogenously added tissue
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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Fig. 2. Structure of platelet-fibrin clots in controls and PV patients. Images from confocal microscope show a structure of PRP-clots derived from blood of healthy controls (A, C) and PV patients (B, D). Fibrin fibers are shown in green and platelet aggregates are seen as an intensive green points surrounded by dense net of thin fibers. Clotting was triggered by recalcification (A, B) or by the addition of TF (C, D). Further details as in Methods. Representative confocal images are shown (n = 12). Magnification bar is 30 μm.
factor on both of the above mentioned processes. As is seen in Fig. 3, tissue factor accelerated clot retraction rate and reduced the speed of fibrinolysis in a dose-dependent manner.
fibrinolysis inversely correlated with CR rate (r = - 0.566; p b 0.001); MCF variable (r = -0.704; p b 0.001), and platelet counts (r = -0.461; p b 0.001).
Effect of aspirin and blocker of platelet GPIIb/IIIa receptors on clot retraction and fibrinolysis in blood from PV patients
Discussion
Fig. 4 shows the results of experiments in which we compared the effect of aspirin and of tirofiban (blocker of platelet GPIIb/IIIa receptors) on the kinetics of CR (panel A) and fibrinolysis speed (panel B) measured in blood from patients suffering from PV. As is seen, aspirin at concentrations of 0.5 mM (two times higher than that used to treat PV patients) failed to affect clot retraction and fibrinolysis. By contrast, tirofiban significantly reduced the clot retraction rate and accelerated the fibrinolysis rate. Correlations between investigated variables The results presented in Table 2 show the correlation of the clot retraction rate constant and platelet count with the speed of fibrinolysis in PV patients. Statistical analysis demonstrates a significant correlation of CR rate with platelet counts (r = 0.546; p b 0.001) but not with erythrocyte concentrations (r = 0.192; p = 0.301). The rate of
The hypothesis linking CR with fibrinolysis is largely based on the observation that the inhibition of clot retraction by blockers of platelet GPIIb/IIIa receptors [7,10,21,22], by cytochalasin D (a platelet actin polymerization inhibitor) [5,7,8], or by inhibitors of platelet energy production [8,9], results in increased speed of clot lysis. Much less is known about the impact of increased CR on the speed of fibrinolysis. Accelerated CR is expected to occur in clinical conditions characterized by enhanced concentrations of platelets in blood. This is because, in the process of platelet-mediated fibrin retraction, platelets provide a contractile apparatus and the active GPIIb/IIIa receptors necessary for generation and transmission of contractile force from inside the cells to connecting fibrin fibers around platelets [1,7,8]. One clinical condition associated with dramatic rises in platelet counts is PV. Platelet counts in PV patients often exceeds 400 × 103 per μl. However, the authors of one study on the lysis resistance of platelet-rich clots from the blood of patients suffering from PV [17], rather excluded the role of clot retraction in the regulation of a
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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Fig. 4. Effect of aspirin and GPIIb/IIIa blocker on the kinetics of clot retraction and lysis of platelet-fibrin clots. Blood samples used for the experiment come from the subset of PV patients not taking aspirin (n = 12). After incubation of samples with ASA (500 μM final conc.) or with tirofiban (200 μg/ml final conc.) the measurements of CR kinetics (A) and fibrinolysis using fluorimetric method (B) were performed as described in Methods. ** p b 0.01; ***p b 0.001. Fig. 3. Effect of increasing TF concentrations on the kinetics of clot retraction (A) and the lysis of platelet-fibrin clots (B). PRP samples (1 ml) were incubated for 2 min at 37 °C. Next, aliquots of 0.4 ml were transferred to plastic tubes containing calcium chloride (2.5 mM final conc.) and tissue factor (Innovin, final conc. as indicated). Plastic tubes were used instead of glass ones to avoid activation of contact pathway of coagulation, thus the only source of thrombin was TF-dependent pathway. Fibrinolysis was evaluated by the addition of Alexa Fluor 488-labeled human fibrinogen to PRP samples. After that, CaCl2 (10 mM final conc.) and TF (final conc. as indicated) were added. Further details in Materials and Methods. *p b 0.05, **p b 0.01.
fibrinolytic activity. This conclusion was based mainly on the observation that lysis velocity of retracted platelet-rich clots from the blood of PV patients and healthy controls were similar. However, in the quoted work clot formation was triggered by relatively high, rather unphysiological thrombin concentrations [17]. The results presented here show that the rate of thrombin generation during platelet-fibrin clot formation (in our experimental model modulated by the addition of increasing TF concentrations) may have a great impact on clot retraction rate and fibrinolysis speed (Fig. 3). In particular, higher TF concentrations (thrombin) are associated with faster clot retraction and reduced fibrinolysis speed [12,23]. It is therefore likely that at high thrombin concentrations, potential differences in the clot retraction rate and fibrinolysis speed between healthy control and PV patients were masked due to the rapid fibrin formation and strong platelet stimulation. Several in vitro studies have demonstrated that the thrombin concentration present at the time of gelation profoundly influences fibrin clot architecture and its resistance to lysis [12,23–25]. Thus, clots formed in the presence of low thrombin concentrations are composed of thick fibrin fibers and are highly susceptible to fibrinolysis; while
clots formed in the presence of high thrombin concentrations are composed of thin fibers and are relatively resistant to fibrinolysis. It is also well accepted that fibrin clots with a high degree of crosslinking and tight fibrin conformation (made of thin fibers) are lysed at a slower rate than those with a loose fibrin conformation (made of thicker fibers). To sum up, it is now clear that the endogenous platelet procoagulant activity and pattern of thrombin generation determine the clot structure, and that the fibrin clot structure, in turn, determines the rate of fibrinolysis [10,11]. Taking this in mind, to better reflect in vivo events in our experimental model we used low TF concentrations to initiate clot formation and fibrinolysis. Recent studies on the kinetics of clot formation in blood from PV patients evaluated by means of thromboelastography indicate that compared with healthy controls, PV patients demonstrated a significant increase in alpha angle parameter [26]. Augmented alpha angle indicates a higher rate of thrombin generation (resulting mainly from the platelet procoagulant response) [27,28] and may imply abnormal dynamics of thrombin formation in the blood of PV patients. At higher thrombin concentrations, fibrin clots with a higher degree of Table 2 Spearman rank correlations between CR and fibrynolysis rates with measured variables in PV patients before ECP. PLT Clot retraction rate Fibrinolysis rate
0.546⁎ -0.461⁎
RBC
MCF
G
CLT
CR rate
0.192 -0.109
0.449⁎ -0.704⁎
0.501⁎ -0.755⁎
0.462⁎ -0.899⁎
-0.566⁎
⁎ p b 0.01.
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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crosslinking and more tight fibrin conformation are likely to be formed [11,24,25]. Taking all this in mind it is highly expected that in PV patients both platelet related generation of thrombin and faster clot retraction may result in formation of greatly crosslinked platelet-fibrin clots that are more resistant to lysis. Consistent with this is the observation reported here that clots formed in blood (PRP) from PV patients, visible in a confocal microscope, demonstrate altered architecture (Fig. 2). In particular, in comparison with healthy controls, clots from PV patients show a distinctly higher degree of crosslinking and possess thinner fibers. Altered clot architecture in PV patients may result not only from faster thrombin formation but also from the abnormal kinetics of clot retraction. The results presented here show that, compared with healthy controls, the rate constant of CR was distinctly higher and final clot volume smaller (Fig. 1), strongly indicating more tight conformation of the platelet fibrin clot. Formation of more tight clots in blood from PV patients confirms also a distinctly enhanced maximum clot firmness (MCF). MCF is a reflection of the absolute strength of the platelet-fibrin clot and is related to the dynamic properties of fibrin, platelet count and the numbers of activated platelet GPIIb/IIIa receptors [27]. Abnormal MCF may result in formation of thrombi that are more stable mechanically and are more resistant to fibrinolysis. In PV patients, augmented CR rate positively correlated with platelet count, but not with erythrocyte concentrations, confirming the crucial role of platelets in the altered kinetics of clot retraction. Lack of correlation of CR with erythrocyte count can be explained by the limited incompressibility of erythrocytes, which has been reported to reduce retraction of the clot [29,30]. In comparison to healthy controls, fibrinolysis speed in PV patients was significantly reduced and was inversely correlated with CR rate (Table 2). The degree of this correlation was significant, strongly indicating the close relationship of these two processes. This is further confirmed by the observation reported here that, in PV patients, fibrinolysis much better correlated with increased retraction rate (r = -0.566; p b 0.001) and MCF (r = -0.704; p b 0.001) than with platelet count (r = -0.461; p b 0.01). This is most likely due to the fact that abnormal retraction is associated both with higher thrombin production (platelet procoagulant response) and augmented contractile force (due to enhanced platelet count) - processes ultimately leading to the formation of highly crosslinked and dense clots resistant to fibrinolysis. To conclude, these data identify CR as an important factor that may contribute to reduced fibrinolysis speed in PV patients. Although the present study emphasizes the role of clot retraction in the altered fibrinolysis speed in PV patients, the contribution of other factors that may account for the reduced fibrinolysis speed in PV patients cannot be excluded. It has been proposed that the elevated plasminogen activator inhibitor type 1 (PAI-1) activity measured in plasma and more active platelet PAI-1 released from activated platelets may serve as a partial explanation for the lysis resistance reported in PV patients [17]. Further quantitative analyses are needed to evaluate the impact of PAI-1 and CR in regulation of fibrinolysis in PV patients. To reduce the risk of thrombotic complications and to improve circulation of the blood by lowering blood viscosity, PV patients routinely undergo cytoreductive treatments. A mainstay in cytoreductive treatment is phlebotomy, which is currently routinely performed in a majority of patients suffering from PV by the use of erythrocytapheresis procedure [31]. ECP procedure enables normalization of the erythrocyte count and blood viscosity but it usually fails to reduce concentration of abnormal platelets. The results reported here indicate that in patients suffering from PV, ECP procedure failed to normalize both clot retraction and fibrinolysis (Fig. 1 and Table 1). The lack of influence of ECP procedure on the CR rate and lysis resistance is most likely due to the low efficacy of ECP in reducing platelet counts. To limit potential platelet-related risk of thrombotic complications in phlebotomized patients, current treatment recommendations
include ECP procedure plus low dose aspirin (ASA) [32,33]. Since about 75% of PV patients included in our studies underwent aspirin treatment, the question arises whether aspirin itself is able to affect clot retraction and fibrinolysis. Unexpectedly, the results presented here clearly indicate that aspirin, even at concentrations two times higher than that routinely used in PV treatment, failed to affect clot retraction and fibrinolysis. In contrast to aspirin, blocking of platelet GPIIb/IIIa receptors by tirofiban resulted in a reduction of abnormal CR and fibrinolysis (Fig. 4). The inefficacy of ASA can be explained by the fact that in our experimental model, platelets were activated by thrombin. It has been reported that in aspirinated platelets activated by thrombin, despite a complete inhibition of cyclooxygenase activity, nearly all of the GPIIb/IIIa receptors, crucial for clot retraction, remained functional [22]. In contrast to our experimental model, under in vivo conditions, i.e. in the blood of PV patients, activation of platelets is more likely to be triggered by stimuli weaker than thrombin i.e. shear forces (related to high viscosity) and by ADP released from damaged erythrocytes. Activation of platelets by low ADP strongly depends on ASA-sensitive thromboxsan A2 production. Appearance of erythrocyte-derived ADP in the blood of PV patients is likely, since in their blood elevated level of free hemoglobin, indicator of erythrocyte damage, have been reported [34]. Thus, our finding does not undermines well documented recommendations for the use of aspirin in antithrombotic treatment of PV patients [32,33]. However, abnormal platelet-related fibrinolysis resistance, may not be normalized by aspirin treatment in the subset of PV patients with concomitant inflammatory state, where TF-induced thrombin formation is likely to occur. Clearly, further studies are needed to substantiate these conclusions. Collectively, the present study shows that in PV patients with thrombocytosis, abnormal CR may result in formation of thrombi that are more resistant to fibrinolysis. ECP procedure failed to normalize both CR rate and fibrinolysis. Unlike GPIIIb/IIIa blockers, aspirin failed to reduce abnormal CR and fibrinolysis resistance in blood from PV patients. Conflict of interest statement We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. References [1] Heemskerk JW, Mattheij NJ, Cosemans JM. Platelet-based coagulation: Different populations, different functions. J Thromb Haemost 2013;11:2–16. [2] Ono A, Westein E, Hsiao S, Nesbitt WS, Hamilton JR, Schoenwaelder SM, et al. Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth. Blood 2008;112:90–9. [3] Tucker KL, Sage T, Gibbins JM. Clot retraction. Methods Mol Biol 2012;788:101–7. [4] Collet JP, Shuman H, Ledger RE, Lee S, Weisel JW. The elasticity of an individual fibrin fiber in a clot. Proc Natl Acad Sci U S A 2005;102:9133–7. [5] Kunitada S, FitzGerald GA, Fitzgerald DJ. Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction. Blood 1992;79:1420–7. [6] Collet JP, Lesty C, Montalescot G, Weisel JW. Dynamic changes of fibrin architecture during fibrin formation and intrinsic fibrinolysis of fibrin-rich clots. J Biol Chem 2003;278:21331–5. [7] Schoenwaelder SM, Ono A, Nesbitt WS, Lim J, Jarman K, Jackson SP. Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces. J Biol Chem 2010;285:2886–96. [8] Misztal T, Przesław K, Rusak T, Tomasiak M. Peroxynitrite-altered platelet mitochondria - a new link between inflammation and hemostasis. Thromb Res 2013;131: e17–25. [9] Misztal T, Rusak T, Tomasiak M. Peroxynitrite may affect clot retraction in human blood through the inhibition of platelet mitochondrial energy production. Thromb Res 2014;133:402–11. [10] Collet JP, Montalescot G, Lesty C, Weisel JW. A structural and dynamic investigation of the facilitating effect of glycoprotein IIb/IIIa inhibitors in dissolving platelet-rich clots. Circ Res 2002;90:428–34. [11] Weisel JW, Litvinov RI. The biochemical and physical process of fibrinolysis and effects of clot structure and stability on the lysis rate. Cardiovasc Hematol Agents Med Chem 2008;6:161–80.
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Regular Article
Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheresis Tomasz Rusak a, Jarosław Piszcz b, Tomasz Misztal a, Justyna Brańska-Januszewska c, Marian Tomasiak a,⁎ a b c
Department of Physical Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland Department of Hematology, Medical University of Bialystok, Skłodowskiej-Curie 24A, 15-276 Bialystok, Poland Department of Biology, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland
a r t i c l e
i n f o
Article history: Received 11 March 2014 Received in revised form 4 April 2014 Accepted 23 April 2014 Available online xxxx Keywords: Clot retraction Clot structure Fibrinolysis Platelets Polycythemia vera Thromboelastometry
a b s t r a c t Using patients with polycythemia vera (PV) as an experimental model, we evaluated the impact of clot retraction (CR) and architecture of the clot on fibrinolysis. We studied the kinetics of clot retraction and the fibrinolysis rate in whole blood from 48 PV patients and 48 healthy controls. Measurements were performed before and after isovolemic eryhrocytapheresis (ECP). CR was measured by optical method. Clot lysis time (CLT) and maximum clot firmness (MCF) were measured by thromboelastometry in recalcified blood supplemented with t-PA and tissue factor. Compared with healthy controls, CR rate in PV patients was higher (0.0219 vs. 0.0138; p b 0.001), the clot volume smaller and MCF elevated (64 vs. 58 mm). CR rate correlated with platelet count (r = 0.546; p = 0.001) but not with erythrocyte concentration (r = 0.192; p N 0.3). Compared with healthy controls, CLT in PV patients was significantly prolonged (158 min vs. 71 min). Fibrinolysis rate inversely correlated with CR rate (r = -0.566; p b 0.001); MCF (r = -0.704; p b 0.001) and platelet count (r = -0.461; p b 0.001). As judged by confocal microscope, in comparison to healthy controls, clots formed in blood from PV patients demonstrated booth a distinctly higher degree of crosslinking and possessed thinner fibers. Altered CR, MCF and fibrinolysis speeds were not normalized following the ECP procedure. Tirofiban (a blocker of platelet GPIIb/IIIa receptors), unlike aspirin, normalized abnormal CR and fibrinolysis in blood from PV patients. Collectively, our data indicate that in PV patients, abnormal CR may result in formation of thrombi that are more resistant to fibrinolysis. ECP and aspirin failed to normalize platelet-related fibrinolysis resistance. © 2014 Elsevier Ltd. All rights reserved.
Introduction Clot retraction is defined as the slow shrinking of a freshly formed platelet-fibrin clot closely attached to the injured blood vessel wall [1–4]. Retraction of the clot makes possible faster recanalization of the occluded (by thrombus) blood vessel which may result in shortening the time of ischemia of neighboring tissues. The retracted clot is more strongly connected with the vessel wall, more stable mechanically, and thus less prone to detaching under conditions of high shear stress [2]. Clot retraction is also proposed to have great impact on the lysis of a freshly formed platelet-fibrin clot. Studies, carried out on models of fibrinolysis and thrombolysis that properly mimic physiological Abbreviations: ASA, aspirin; CR, clot retraction; CLT, clot lysis time; ECP, erythracytapheresis procedure; MCF, maximum clot firmness; PAI-1, plasminogen activator inhibitor-1; PV, polycythemia vera; PRP, platelet rich plasma; RBC, red blood cells; TF, tissue factor; tPA, tissue plasminogen activator; WBC, white blood cells. ⁎ Corresponding author. Tel.: +48 85 748 5714; fax: +48 85 748 5416. E-mail addresses: [email protected] (T. Rusak), [email protected] (J. Piszcz), [email protected] (T. Misztal), [email protected] (J. Brańska-Januszewska), [email protected] (M. Tomasiak).
conditions [2,5–7], strongly indicate that platelets make clots resistant to lysis. This phenomenon has been termed “platelet-mediated fibrinolysis resistance”. The mechanism(s) by which human platelets make clots resistant to fibrinolysis are not completely understood, but the accumulated evidence points to clot retraction as one of the major causes. Understanding the cross-talk between clot retraction and fibrinolysis is of great clinical importance since reactive nitrogen species released by activated inflammatory cells have recently been reported to modulate clot retraction rate [8,9]. Although the link between clot retraction and fibrinolysis velocity in vitro is well documented [10–12], much less is known about a similar relationship in clinical conditions characterized with altered hemostasis. A prominent example of such a condition is polycythemia vera. Polycythemia vera (PV) is a chronic myeloproliferative neoplasm, characterized by increased red cell mass and is often accompanied by thrombocytosis and leukocytosis [13–15]. The clinical course of the disease can be complicated by both thrombotic and hemorrhagic events which remain the leading causes of morbidity and mortality in untreated PV patients [15,16]. What’s more, both altered clot retraction and fibrinolysis have been reported in PV patients [17,18].
http://dx.doi.org/10.1016/j.thromres.2014.04.025 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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Consequently, this study was undertaken to evaluate the link between clot retraction and fibrinolysis in blood from patients suffering from PV. Another aim was to determine whether normalization of the erythrocyte count by means of routinely performed isovolemic erythrocytapheresis (ECP) affects clot retraction and fibrinolysis in patients suffering from PV. Materials and methods Study subjects Forty eight patients (18 men, 30 women) with PV, diagnosed according to criteria established by the World Health Organization [19], and an age-matched control group of 48 healthy volunteers (19 men, 29 women) with normal blood cell counts were entered into the study. Thirty six PV patients took low dose aspirin, whereas 12 of them discontinued aspirin therapy at least one week before blood collection. At the moment of blood collection, patients enrolled in the study did not undergo pharmacological cytoreductive therapy. Healthy volunteers had not taken medication known to affect platelet function and/or coagulation for at least 10 days before blood sampling. None of the study subjects had taken oral contraceptives or hormone replacement therapy for at least two months before blood collection. The study protocol was approved by the Ethics Committee at the Medical University of Bialystok. The procedures were in accordance with the Declaration of Helsinki of 1975, as revised in 2000 and blood samples were obtained with the subjects’ informed consent. Blood collection and erythrocytapheresis procedure Venous blood was collected with minimum trauma and stasis via a 21-gauge needle (0.8 × 40 mm) into 9 ml polypropylene vacuum tubes (Vacuette, Greiner Bio-One, Kremsmünster, Austria) containing 130 mM trisodium citrate. All PV patients were undergoing erythrocytapheresis (ECP), which was carried out using MCS + cell separator (Haemonetics, Braintree, MA, USA). The removed erythrocyte volume (100-410 ml) was replaced with an equal amount of physiological saline as fluid compensation. The instrument has software which, based on the initial hematocrit, body weight and sex of the subject, precisely determines the blood volume and hematocrit at the end of the procedure. Blood was collected 20 minutes before ECP and an hour after the procedure.
Evaluation of platelet-fibrin clot lysis by fluorimetric method Lysis of platelet-fibrin clots was estimated according to Boulaftali et al. with some modifications [20]. PRP samples (0.4 ml) were supplemented with Alexa Fluor 488-labeled human fibrinogen (final conc. 75 μg/ml) and incubated at 37 °C for 2 minutes. Afterwards, aliquots of 0.1 ml were transferred to plastic tubes containing 0.775 ml of Tyrode-Hepes buffer supplemented with CaCl2 (final conc. 10 mM) and tissue factor (Innovin, final conc. as indicated), stirred for 30 sec and incubated at 37 °C for 1 hour in the dark to allow full clot retraction. To perform lysis, the retracted clots were transferred to polypropylene tubes containing a 1.5 ml of fresh T-H buffer and incubated at 37 °C for 24 hours in the dark. Then, samples were centrifuged (5 min, 11000 ×g, room temperature) to remove clots, and the aliquots (500 μl) of supernatant were collected for determination of fluorescence intensity (Ex 488 nm, Em 522 nm) associated with Alexa Fluor 488containing fibrin degradation products (AF488-FDP), using the Hitachi F-7000 fluorimeter (Hitachi Ltd., Japan). Fluorescence intensity reflects the progress of fibrinolysis. To determine the maximal fibrinolysis (100% lysis) the retracted clots were incubated 37 °C for 24 hours in the dark with tPA added to the final concentration of 1 μg/ml. In some experiments, PRP samples were incubated with aspirin (final conc. 500 μM) or tirofiban (final conc. 200 μg/ml) for 10 min before the addition of Alexa Fluor 488-fibrinogen. Confocal microscopy of platelet-fibrin clots Samples (100 μl) of PRP were supplemented with Alexa Fluor 488labeled human fibrinogen (0.15 μM final conc., approximately 15 dye molecules for each fibrinogen molecule) and preincubated for 2 min. at 37 °C. Next, CaCl2 (20 mM final conc.) or CaCl2 + recombinant tissue factor (10 mM and 140 ng/ml final conc. respectively) were added, mixed vigorously and aliquots of 25 μL were transferred to microchamber slides (Ibidi μ-slide VI; Thistle Scientific). Clotting samples were incubated for 2 h at 37 °C in humid atmosphere, protected from light. After gelation time, clots architecture was imaged using fluorescence microscope with confocal imaging system – Nikon ECLIPSE Ti/C1 Plus (Ex 488 nm/Em 515/30 nm, 100× magnification). The images were acquired with a field of view of 120 × 120 μm. At least 10 pictures of different areas of each clot was taken and one representative image is presented.
Measurement of kinetics of clot retraction Routine hematological assays Measurement of the kinetics of clot retraction in whole blood were performed in non-siliconized glass tubes as described before [8]. Pictures were taken for one hour at 10 min intervals and after 120 min using a digital camera. Quantification of retraction was performed by assessment of the clot area by use of Motic Images Plus 2.0 ML software, and data were processed using Microsoft Excel. Clot surface areas were plotted as a percentage of maximal retraction (i.e. volume of platelet suspension). Data were expressed as follows: percentage of retraction (relative clot volume) = (area t0 − area t)/(area t0) × 100. Kinetics of clot retraction was characterized by the calculation of rate constant of retraction process. Thromboelastometric analyses Thromboelastometric measurements were performed on the ROTEM® system (Tem International GmbH, Manheim, Germany). Recalcified (10 mM CaCl2) blood was assessed for fibrinolytic potential using either 140 ng/ml tissue factor (TF), and 125 ng/ml of tissue plasminogen activator (tPA). We measured the parameters characterized clot strength (maximal clot firmness) and fibrinolysis (percentage reduction of MCF in time, clot lysis time). The ROTEM analysis was started 30 min after the blood was drawn by adding 320 μl of blood to a cuvette already containing 20 μl of re-calcification reagents.
Complete blood cell count, Hct and hemoglobin levels, were measured by an automated hematology analyzer (Sysmex SE9000, Toa Medical Electronics, Kobe, Japan). Data analysis Data reported in this paper are the median of the number of determinations indicated (n). Statistical analysis was performed by Mann-Whitney-Wilcoxon U test and elaboration of experimental data by the use of STATISTICA software (StatSoft, Tulsa, OK, USA). Differences were considered significant at a p value b 0.05. Correlations were assessed by a non-parametric test (Spearman’s rank correlation coefficient, r). Results Erythrocytapheresis-evoked changes in blood morphology The results presented in Table 1 show that the erythrocyte (RBC), leukocyte (WBC) and platelet counts of patients with PV were significantly greater than those of the control subjects. Erythrocytapheresis (ECP) resulted in a significant reduction in erythrocyte count, hematocrit and
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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Table 1 Characteristics of study subjects. Polycythemia vera (n = 48)
men/woman Age, median (range) RBC (×1012/l) WBC (×109/l) Platelets (×109/l) Haematocrit Hemoglobin (g/dl) Thromboelastometry: G (dynes/cm2) MCF (mm) LI30 (%): LI60 (%): LI90 (%): CLT (min):
Controls (n = 48)
before ECP
after ECP
18/30 58.9 (43 ÷ 75) 6.04 (4.51 ÷ 9.07) ⁎⁎ 10.0 (6.0 ÷ 23.3) ⁎⁎ 394 (183 ÷ 792)⁎ 0.533 (.465 ÷ .627) ⁎⁎ 17.3 (14.6 ÷ 22.4)⁎⁎
5.36 (4.07 ÷ 7.21) ⁎, † 9.1 (4.6 ÷ 21.1)⁎ ⁎ 385 (182 ÷ 755)⁎ 0.467 (.402 ÷ .545)⁎, † 15.2 (12.4 ÷ 19.4) ⁎,†
19/29 57.2 (42 ÷ 71) 4.48 (4.03 ÷ 5.84) 6.3 (3.6 ÷ 10.9) 219 (115 ÷ 351) 0.412 (.358 ÷ .507) 13.7 (12.2 ÷ 17.0)
8973 (4636 ÷ 16408)⁎ 64 (51 ÷ 76)⁎ 0 (0 ÷ 5) ⁎ 11 (2 ÷ 49) ⁎⁎ 29 (6 ÷ 93) ⁎⁎ 158 (84 ÷ 268) ⁎
9426 (4898 ÷ 16376) ⁎ 65 (51 ÷ 74) ⁎ 0 (0 ÷ 3) ⁎ 10 (2 ÷ 55) ⁎⁎ 26 (8 ÷ 94) ⁎⁎ 156 (93 ÷ 242) ⁎
7001(3043 ÷ 10454) 58 (39 ÷ 68) 6 (1 ÷ 49) 93 (29 ÷ 100) 100 (58 ÷ 100) 71 (49 ÷ 116)
⁎) p b 0.05; ⁎⁎) p b 0.01 (compared with healthy subjects); ) p b 0.05 (compared with PV patients before ECP). G – the absolute shear elastic modulus of the sample is then calculated from the maximal clot firmness (MCF) as follows: G = 5000*MCF/(100-MCF). LI (Lysis Index) - a reduction of amplitude relative to MCF at 30, 60 and 90 min after the initiation of blood clotting. CLT – clot lysis time (time of complete fibrinolysis). †
hemoglobin values. The procedure practically was without any effect on platelet count which remained significantly higher than those in healthy controls.
Kinetics of clot retraction, effect of erythrocytapheresis Fig. 1 shows the results of experiments in which we compared the kinetics of CR (panel A) and the volume of retracted clots (panel B) in the blood of both PV patients and healthy control. As is seen, the rate constant of clot retraction was significantly higher in PV patients than in healthy controls (0.0219 vs. 0.0138 min-1; p b 0.001). Correspondingly compared with healthy subjects, PV patients have strongly reduced volume of retracted clots measured 40 minutes after initiation of clotting (0.44 vs. 0.56). ECP procedure failed to normalize the kinetics of clot retraction (k = 0.0217 min-1) and the volume of retracted clots (0.45) in PV patients. Fibrinolysis resistance evaluated by ROTEM rotational tromboelastometry Experiments shown in Table 1 were performed to evaluate the lysis speed of clots formed in blood from PV patients and healthy controls. The fibrinolytic response by tPA was assessed with the use of ROTEM software, thereby providing the lysis progress at 30, 60, and 90 minutes, complete lysis time, MCF and G variables. As is shown, compared to healthy controls, polycythemic patients have prolonged lysis times (158 vs. 71 min; p b 0.001) and augmented MCF (64 vs. 58 mm) and G (8973 vs. 7001 dynes/cm2; p b 0.001) variables, indicating the existence of a fibrinolysis resistance state. Neither MCF variable (65 mm) nor clot lysis time (156 min) were markedly changed following the erythrocytapheresis procedure, indicating its inefficacy in reducing fibrinolysis resistance. Architecture of platelet fibrin clots The results presented in Fig. 2 show the architecture of plateletfibrin clots formed in blood from healthy controls (panel A, C) and from PV patients (panel B, D) visualized with confocal microscope. As is seen, the fibrin network architecture of clots formed in blood from PV and from healthy controls dramatically differs. More specifically, compared with healthy subjects, PV patients have clots that are much more dense, more crosslinked, and are made from thinner fibers.
Fig. 1. Clot retraction in healthy subjects (control; n = 48) and PV patients (n = 48; before and after ECP procedure). Clot surface areas were assessed by digital processing and plotted as percentage of volume of blood suspension. Panel A shows volumes of clots measured 40 minutes after initiation of clotting. Panel B represents the clot retraction rates (to each patient) calculated from the slope of the clot volume against time (min-1).
Impact of thrombin formation rate on clot retraction and fibrinolysis speed To evaluate whether a pattern of thrombin generation affects clot retraction and fibrinolysis speed, in our experimental model we tested the impact of increasing concentrations of exogenously added tissue
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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Fig. 2. Structure of platelet-fibrin clots in controls and PV patients. Images from confocal microscope show a structure of PRP-clots derived from blood of healthy controls (A, C) and PV patients (B, D). Fibrin fibers are shown in green and platelet aggregates are seen as an intensive green points surrounded by dense net of thin fibers. Clotting was triggered by recalcification (A, B) or by the addition of TF (C, D). Further details as in Methods. Representative confocal images are shown (n = 12). Magnification bar is 30 μm.
factor on both of the above mentioned processes. As is seen in Fig. 3, tissue factor accelerated clot retraction rate and reduced the speed of fibrinolysis in a dose-dependent manner.
fibrinolysis inversely correlated with CR rate (r = - 0.566; p b 0.001); MCF variable (r = -0.704; p b 0.001), and platelet counts (r = -0.461; p b 0.001).
Effect of aspirin and blocker of platelet GPIIb/IIIa receptors on clot retraction and fibrinolysis in blood from PV patients
Discussion
Fig. 4 shows the results of experiments in which we compared the effect of aspirin and of tirofiban (blocker of platelet GPIIb/IIIa receptors) on the kinetics of CR (panel A) and fibrinolysis speed (panel B) measured in blood from patients suffering from PV. As is seen, aspirin at concentrations of 0.5 mM (two times higher than that used to treat PV patients) failed to affect clot retraction and fibrinolysis. By contrast, tirofiban significantly reduced the clot retraction rate and accelerated the fibrinolysis rate. Correlations between investigated variables The results presented in Table 2 show the correlation of the clot retraction rate constant and platelet count with the speed of fibrinolysis in PV patients. Statistical analysis demonstrates a significant correlation of CR rate with platelet counts (r = 0.546; p b 0.001) but not with erythrocyte concentrations (r = 0.192; p = 0.301). The rate of
The hypothesis linking CR with fibrinolysis is largely based on the observation that the inhibition of clot retraction by blockers of platelet GPIIb/IIIa receptors [7,10,21,22], by cytochalasin D (a platelet actin polymerization inhibitor) [5,7,8], or by inhibitors of platelet energy production [8,9], results in increased speed of clot lysis. Much less is known about the impact of increased CR on the speed of fibrinolysis. Accelerated CR is expected to occur in clinical conditions characterized by enhanced concentrations of platelets in blood. This is because, in the process of platelet-mediated fibrin retraction, platelets provide a contractile apparatus and the active GPIIb/IIIa receptors necessary for generation and transmission of contractile force from inside the cells to connecting fibrin fibers around platelets [1,7,8]. One clinical condition associated with dramatic rises in platelet counts is PV. Platelet counts in PV patients often exceeds 400 × 103 per μl. However, the authors of one study on the lysis resistance of platelet-rich clots from the blood of patients suffering from PV [17], rather excluded the role of clot retraction in the regulation of a
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
T. Rusak et al. / Thrombosis Research xxx (2014) xxx–xxx
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Fig. 4. Effect of aspirin and GPIIb/IIIa blocker on the kinetics of clot retraction and lysis of platelet-fibrin clots. Blood samples used for the experiment come from the subset of PV patients not taking aspirin (n = 12). After incubation of samples with ASA (500 μM final conc.) or with tirofiban (200 μg/ml final conc.) the measurements of CR kinetics (A) and fibrinolysis using fluorimetric method (B) were performed as described in Methods. ** p b 0.01; ***p b 0.001. Fig. 3. Effect of increasing TF concentrations on the kinetics of clot retraction (A) and the lysis of platelet-fibrin clots (B). PRP samples (1 ml) were incubated for 2 min at 37 °C. Next, aliquots of 0.4 ml were transferred to plastic tubes containing calcium chloride (2.5 mM final conc.) and tissue factor (Innovin, final conc. as indicated). Plastic tubes were used instead of glass ones to avoid activation of contact pathway of coagulation, thus the only source of thrombin was TF-dependent pathway. Fibrinolysis was evaluated by the addition of Alexa Fluor 488-labeled human fibrinogen to PRP samples. After that, CaCl2 (10 mM final conc.) and TF (final conc. as indicated) were added. Further details in Materials and Methods. *p b 0.05, **p b 0.01.
fibrinolytic activity. This conclusion was based mainly on the observation that lysis velocity of retracted platelet-rich clots from the blood of PV patients and healthy controls were similar. However, in the quoted work clot formation was triggered by relatively high, rather unphysiological thrombin concentrations [17]. The results presented here show that the rate of thrombin generation during platelet-fibrin clot formation (in our experimental model modulated by the addition of increasing TF concentrations) may have a great impact on clot retraction rate and fibrinolysis speed (Fig. 3). In particular, higher TF concentrations (thrombin) are associated with faster clot retraction and reduced fibrinolysis speed [12,23]. It is therefore likely that at high thrombin concentrations, potential differences in the clot retraction rate and fibrinolysis speed between healthy control and PV patients were masked due to the rapid fibrin formation and strong platelet stimulation. Several in vitro studies have demonstrated that the thrombin concentration present at the time of gelation profoundly influences fibrin clot architecture and its resistance to lysis [12,23–25]. Thus, clots formed in the presence of low thrombin concentrations are composed of thick fibrin fibers and are highly susceptible to fibrinolysis; while
clots formed in the presence of high thrombin concentrations are composed of thin fibers and are relatively resistant to fibrinolysis. It is also well accepted that fibrin clots with a high degree of crosslinking and tight fibrin conformation (made of thin fibers) are lysed at a slower rate than those with a loose fibrin conformation (made of thicker fibers). To sum up, it is now clear that the endogenous platelet procoagulant activity and pattern of thrombin generation determine the clot structure, and that the fibrin clot structure, in turn, determines the rate of fibrinolysis [10,11]. Taking this in mind, to better reflect in vivo events in our experimental model we used low TF concentrations to initiate clot formation and fibrinolysis. Recent studies on the kinetics of clot formation in blood from PV patients evaluated by means of thromboelastography indicate that compared with healthy controls, PV patients demonstrated a significant increase in alpha angle parameter [26]. Augmented alpha angle indicates a higher rate of thrombin generation (resulting mainly from the platelet procoagulant response) [27,28] and may imply abnormal dynamics of thrombin formation in the blood of PV patients. At higher thrombin concentrations, fibrin clots with a higher degree of Table 2 Spearman rank correlations between CR and fibrynolysis rates with measured variables in PV patients before ECP. PLT Clot retraction rate Fibrinolysis rate
0.546⁎ -0.461⁎
RBC
MCF
G
CLT
CR rate
0.192 -0.109
0.449⁎ -0.704⁎
0.501⁎ -0.755⁎
0.462⁎ -0.899⁎
-0.566⁎
⁎ p b 0.01.
Please cite this article as: Rusak T, et al, Platelet-related fibrinolysis resistance in patients suffering from PV. Impact of clot retraction and isovolemic erythrocytapheres, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.025
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T. Rusak et al. / Thrombosis Research xxx (2014) xxx–xxx
crosslinking and more tight fibrin conformation are likely to be formed [11,24,25]. Taking all this in mind it is highly expected that in PV patients both platelet related generation of thrombin and faster clot retraction may result in formation of greatly crosslinked platelet-fibrin clots that are more resistant to lysis. Consistent with this is the observation reported here that clots formed in blood (PRP) from PV patients, visible in a confocal microscope, demonstrate altered architecture (Fig. 2). In particular, in comparison with healthy controls, clots from PV patients show a distinctly higher degree of crosslinking and possess thinner fibers. Altered clot architecture in PV patients may result not only from faster thrombin formation but also from the abnormal kinetics of clot retraction. The results presented here show that, compared with healthy controls, the rate constant of CR was distinctly higher and final clot volume smaller (Fig. 1), strongly indicating more tight conformation of the platelet fibrin clot. Formation of more tight clots in blood from PV patients confirms also a distinctly enhanced maximum clot firmness (MCF). MCF is a reflection of the absolute strength of the platelet-fibrin clot and is related to the dynamic properties of fibrin, platelet count and the numbers of activated platelet GPIIb/IIIa receptors [27]. Abnormal MCF may result in formation of thrombi that are more stable mechanically and are more resistant to fibrinolysis. In PV patients, augmented CR rate positively correlated with platelet count, but not with erythrocyte concentrations, confirming the crucial role of platelets in the altered kinetics of clot retraction. Lack of correlation of CR with erythrocyte count can be explained by the limited incompressibility of erythrocytes, which has been reported to reduce retraction of the clot [29,30]. In comparison to healthy controls, fibrinolysis speed in PV patients was significantly reduced and was inversely correlated with CR rate (Table 2). The degree of this correlation was significant, strongly indicating the close relationship of these two processes. This is further confirmed by the observation reported here that, in PV patients, fibrinolysis much better correlated with increased retraction rate (r = -0.566; p b 0.001) and MCF (r = -0.704; p b 0.001) than with platelet count (r = -0.461; p b 0.01). This is most likely due to the fact that abnormal retraction is associated both with higher thrombin production (platelet procoagulant response) and augmented contractile force (due to enhanced platelet count) - processes ultimately leading to the formation of highly crosslinked and dense clots resistant to fibrinolysis. To conclude, these data identify CR as an important factor that may contribute to reduced fibrinolysis speed in PV patients. Although the present study emphasizes the role of clot retraction in the altered fibrinolysis speed in PV patients, the contribution of other factors that may account for the reduced fibrinolysis speed in PV patients cannot be excluded. It has been proposed that the elevated plasminogen activator inhibitor type 1 (PAI-1) activity measured in plasma and more active platelet PAI-1 released from activated platelets may serve as a partial explanation for the lysis resistance reported in PV patients [17]. Further quantitative analyses are needed to evaluate the impact of PAI-1 and CR in regulation of fibrinolysis in PV patients. To reduce the risk of thrombotic complications and to improve circulation of the blood by lowering blood viscosity, PV patients routinely undergo cytoreductive treatments. A mainstay in cytoreductive treatment is phlebotomy, which is currently routinely performed in a majority of patients suffering from PV by the use of erythrocytapheresis procedure [31]. ECP procedure enables normalization of the erythrocyte count and blood viscosity but it usually fails to reduce concentration of abnormal platelets. The results reported here indicate that in patients suffering from PV, ECP procedure failed to normalize both clot retraction and fibrinolysis (Fig. 1 and Table 1). The lack of influence of ECP procedure on the CR rate and lysis resistance is most likely due to the low efficacy of ECP in reducing platelet counts. To limit potential platelet-related risk of thrombotic complications in phlebotomized patients, current treatment recommendations
include ECP procedure plus low dose aspirin (ASA) [32,33]. Since about 75% of PV patients included in our studies underwent aspirin treatment, the question arises whether aspirin itself is able to affect clot retraction and fibrinolysis. Unexpectedly, the results presented here clearly indicate that aspirin, even at concentrations two times higher than that routinely used in PV treatment, failed to affect clot retraction and fibrinolysis. In contrast to aspirin, blocking of platelet GPIIb/IIIa receptors by tirofiban resulted in a reduction of abnormal CR and fibrinolysis (Fig. 4). The inefficacy of ASA can be explained by the fact that in our experimental model, platelets were activated by thrombin. It has been reported that in aspirinated platelets activated by thrombin, despite a complete inhibition of cyclooxygenase activity, nearly all of the GPIIb/IIIa receptors, crucial for clot retraction, remained functional [22]. In contrast to our experimental model, under in vivo conditions, i.e. in the blood of PV patients, activation of platelets is more likely to be triggered by stimuli weaker than thrombin i.e. shear forces (related to high viscosity) and by ADP released from damaged erythrocytes. Activation of platelets by low ADP strongly depends on ASA-sensitive thromboxsan A2 production. Appearance of erythrocyte-derived ADP in the blood of PV patients is likely, since in their blood elevated level of free hemoglobin, indicator of erythrocyte damage, have been reported [34]. Thus, our finding does not undermines well documented recommendations for the use of aspirin in antithrombotic treatment of PV patients [32,33]. However, abnormal platelet-related fibrinolysis resistance, may not be normalized by aspirin treatment in the subset of PV patients with concomitant inflammatory state, where TF-induced thrombin formation is likely to occur. Clearly, further studies are needed to substantiate these conclusions. Collectively, the present study shows that in PV patients with thrombocytosis, abnormal CR may result in formation of thrombi that are more resistant to fibrinolysis. ECP procedure failed to normalize both CR rate and fibrinolysis. Unlike GPIIIb/IIIa blockers, aspirin failed to reduce abnormal CR and fibrinolysis resistance in blood from PV patients. Conflict of interest statement We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. References [1] Heemskerk JW, Mattheij NJ, Cosemans JM. Platelet-based coagulation: Different populations, different functions. J Thromb Haemost 2013;11:2–16. [2] Ono A, Westein E, Hsiao S, Nesbitt WS, Hamilton JR, Schoenwaelder SM, et al. Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth. Blood 2008;112:90–9. [3] Tucker KL, Sage T, Gibbins JM. Clot retraction. Methods Mol Biol 2012;788:101–7. [4] Collet JP, Shuman H, Ledger RE, Lee S, Weisel JW. The elasticity of an individual fibrin fiber in a clot. Proc Natl Acad Sci U S A 2005;102:9133–7. [5] Kunitada S, FitzGerald GA, Fitzgerald DJ. Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction. Blood 1992;79:1420–7. [6] Collet JP, Lesty C, Montalescot G, Weisel JW. Dynamic changes of fibrin architecture during fibrin formation and intrinsic fibrinolysis of fibrin-rich clots. J Biol Chem 2003;278:21331–5. [7] Schoenwaelder SM, Ono A, Nesbitt WS, Lim J, Jarman K, Jackson SP. Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces. J Biol Chem 2010;285:2886–96. [8] Misztal T, Przesław K, Rusak T, Tomasiak M. Peroxynitrite-altered platelet mitochondria - a new link between inflammation and hemostasis. Thromb Res 2013;131: e17–25. [9] Misztal T, Rusak T, Tomasiak M. Peroxynitrite may affect clot retraction in human blood through the inhibition of platelet mitochondrial energy production. Thromb Res 2014;133:402–11. [10] Collet JP, Montalescot G, Lesty C, Weisel JW. A structural and dynamic investigation of the facilitating effect of glycoprotein IIb/IIIa inhibitors in dissolving platelet-rich clots. Circ Res 2002;90:428–34. [11] Weisel JW, Litvinov RI. The biochemical and physical process of fibrinolysis and effects of clot structure and stability on the lysis rate. Cardiovasc Hematol Agents Med Chem 2008;6:161–80.
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