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Thrombin generation in clinical conditions
Thrombosis Research 129 (2012) 367–370
Contents lists available at SciVerse ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Mini Review
Thrombin generation in clinical conditions Hugo ten Cate ⁎ Dept. of Internal medicine, laboratory of Clinical Thrombosis and Haemostasis, and Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands
a r t i c l e
i n f o
Article history: Received 6 October 2011 Received in revised form 6 October 2011 Accepted 18 October 2011 Available online 12 November 2011 Keywords: Thrombin generation Venous thromboembolism Arterial thrombosis-atherosclerosis Bleeding
a b s t r a c t Commercial assays for determining thrombin generation in plasma are being tested in clinical conditions associated with thrombosis or bleeding. While pre-analytical conditions remain a source of inter laboratory variation, demanding for further standardization, clinical research proceeds. In patients at risk of venous thrombosis thrombin generation (TG) analysis may be utilized to detect underlying thrombophilia and this has been achieved both with addition of thrombomodulin or activated protein C, to test the contribution of the protein C system. In patients with documented venous thromboembolism, increased TG values are seen in those patients at greatest risk for recurrence, although the data are not consistent yet. In patients with arterial vascular disease, effects on TG patterns are seen that both reflect atherosclerosis (and its risk factors) and link to risk of recurrent atherothrombosis (coronary or stroke), but the data are limited. In patients with a bleeding diathesis, like hemophilia, the main importance of TG assays lies in the application for monitoring replacement therapy, either with factor concentrate or rFVIIa. An interesting application is in conjunction with thromboelastography, for monitoring peri-operative transfusion policy. Finally, TG analysis may contribute to monitoring anticoagulant drug treatment, but these and other applications would greatly benefit from whole blood, point of care applications of TG testing. © 2011 Published by Elsevier Ltd.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Methodology . . . . . . . . . . . . . . . . . . . . . . . . . Utilizing thrombin generation analysis; studies on venous thrombosis Studies on arterial vascular disease . . . . . . . . . . . . . . Studies on bleeding . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future developments . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction Thrombin is a key enzyme in the blood coagulation cascade combining pro- and anticoagulant functions through controlled
Abbreviations: TG, thrombin generation; PPP, platelet poor plasma; SSC, Scientific Subcommittee of the International Society for Thrombosis and Haemostasis; VTE, venous thromboembolism; MI, myocardial infarction; APTT, activated partial trhomboplastin time. ⁎ Laboratory of Clinical Thrombosis and Haemostasis, Maastricht University Medical Center, Universiteitssingel 50, PO Box 616, 6200 MD Maastricht. Tel.: + 31 43 3884262; fax: + 31 43 3884159. E-mail address: [email protected]. 0049-3848/$ – see front matter © 2011 Published by Elsevier Ltd. doi:10.1016/j.thromres.2011.10.017
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regulation of the cascade activity [1]. Either by direct limited proteolysis of other coagulation proteins, yielding active enzymes like factor Xa, or by interacting with cellular receptors like thrombomodulin or protease activated receptors (PARs), thrombin displays multiple functions in the vasculature [2]. Because of its central position the formation of thrombin is considered one of the most important steps in coagulation. For this reason it is not surprising that investigators have tried since many years to study its formation in plasma based systems [3]. Finally, commercial methods for studying thrombin generation (TG) in plasma have now become available, allowing for an expanding number of studies on TG among the research community. In this concise review I will discuss some of the current developments in TG analysis and testing in clinical studies and try to
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H. ten Cate / Thrombosis Research 129 (2012) 367–370
delineate the perspective for using this methodology in the clinical arena. Methodology There are different approaches to the measurement of thrombin formation. An established and commercialized approach is the use of immunoassays, usually Elisa's, to quantify the activation peptide released from prothrombin upon activation by factor Xa (F1 + 2 fragment). The other Elisa approach is aimed at the thrombin antithrombin (TAT) complex. Both methods give an estimate of the amount of thrombin formed in blood at a given moment in time. Many clinical studies have employed these assays to study TG in vivo. The other approach is to quantify the amount of thrombin that can be generated ex vivo by the addition of a small amount of trigger (usually tissue factor) to a plasma sample, to assess the potential to generate thrombin, hence the name of “endogenous thrombin potential” for this type of analysis. Since both approaches are sensitive to low amounts of tissue factor and also to contact activation occurring in the course of blood sampling and processing, the quality of the plasma sample is an important preanalytical factor. For the activation peptide assays, pioneering studies by Rosenberg and Bauer on the prethrombotic state [4] considered preanalytical aspects, including the quality of the blood sample (based on degree of hemolysis etc.), but they employed radioimmunoassays. For the commercial F1 + 2 and TAT elisa's there is no good evidence suggesting specific procedures necessary other than obtaining a properly collected citrated plasma sample. This narrative review paper mainly deals with the capacity assays, in which TG is measured as thrombin potential. Several issues including the type of blood sampling device (Vacutainer® or Butterfly®), the test tube, citrate concentration and the plasma preparation have been investigated and reported [5]. One important and somewhat controversial factor is the need to prevent contact activation. While the addition of corn trypsin inhibitor (CTI) has been advocated for this purpose, CTI is expensive and only acts when already present in the test tube. In our hands, CTI was not required at tissue factor concentrations > 0.5 pmoles, however this was only tested in normal plasma, not in patients [5]. The analysis of thrombin generation is usually done with platelet poor plasma (PPP), although the method can be modified to test platelet rich plasma. The application on PPP also allows for measurement in frozen-thawed plasma from clinical studies, although there is little information about the effects of storage on the quality of the sample. When tested batchwise and including normal plasma to control for technical variation, not more than a random effect of freezing-thawing, would need to be expected. Three commercial techniques have sofar been described: the Calibrated Automated Thrombogram (Trombinoscope/Stago), the Thrombin Generation Test (Siemens) and Technothrombin TGA (Technoclone). The techniques differ considerably in terms of (concentrations of) reagents, including the use of either fluorogenic or chromogenic substrates. Although at fixed test conditions these assays may perform well within one center, comparison of these methods applied on test samples among different centers yielded major variations [6]. It therefore remains of utmost importance to clearly define test conditions, preferably using standard, reference plasma for normalization of TG values. The SSC should take the lead in this process. In the absence of such recommendations or guidelines, investigators best follow the procedures recommended by the manufacturers, at least for testing of clinical samples, in order to minimize the degree of variation imposed when homemade phospholipids, different brands of tissue factor etcetera are applied. Utilizing thrombin generation analysis; studies on venous thrombosis Analyzing the potential to form thrombin (ETP) assumes that it reflects the potential of the patient to form thrombosis, under
thrombogenic conditions like surgery or malignancy. Typically, this relationship between thrombin and thrombosis would be anticipated for venous thromboembolism (VTE), while given the lower impact of hypercoagulability in arterial thrombosis, it is a priori doubtful, whether ETP would also reflect the risk of arterial thrombosis, certainly when tested in PPP, without platelets. In subjects at risk of VTE (thrombophilia), TG analysis may add to the diagnostic workup, being able to detect intermediate thrombophilic phenotypes [6–8]. This will however require TG analysis also with the addition of thrombomodulin or activated protein C, in order to test both the basic TG and TG after full activation of the protein C system. In population based studies Lutsey et al. demonstrated an increased risk for VTE (primarily idiopathic) for elevated peak values of TG [9]. Tripodi and colleagues showed that clinical assessment of VTE risk in low, moderate or high was reflected in TG values when determined in the presence of thrombomodulin [10]. More specifically, modifications of thrombin generation analysis have been employed over the past decades to characterize both genetic and acquired thrombophilia, attributable to factor V Leiden and oral contraceptives respectively [7]. Before TG analysis can be applied in the workup of individual patients (thrombophilia screening) this will require extensive validation of test conditions, including assessment of reference values, within the routine laboratory. In patients with suspected VTE, thrombin generation analysis may help to identify low risk subjects [11], although the use of this test in the acute phase of disease may be prone to acute phase effects of depletion of clotting factors due to thrombosis that may obscure the test interpretation. Rather, in this situation, d-dimer testing may remain the first choice test for excluding thrombosis. In patients with documented VTE several studies show a predicted relationship between thrombin generation and risk of VTE. The first study by Hron et al., showed a low risk of recurrent VTE in patients with a TG value below a certain cutoff level [12]. Increased TG was associated with an increased risk of recurrent VTE in studies from Tripodi et al. [13] and Besser et al. [14], however this was not confirmed by van Hylckama Vlieg et al. finding a risk association with a first, but not with recurrent VTE events [15]. Following a first deep venous thrombosis, patients show persistently elevated TG during 24 months follow up, with reduced response to thrombomodulin, as compared to controls [16]. Whether this persistent hypercoagulability adds to single measurements of TG, to predict recurrence after cessation of anticoagulation, remains to be tested. TG analysis may be of interest in specific groups of patients at variable but elevated risk of venous thrombosis, such as those with malignancies. In the large Vienna Cancer and Thrombosis Study elevated thrombin peak values (defined as values ≥ 611 nM, being the 75th percentile) were associated with an increased risk of VTE with a hazard ratio of 2.1 (95% CI 1.3-3.3) in multivariate analysis [17]. Similarly, TG may predict increased risk of VTE in cancer patients during chemotherapy [18]. In specific malignant disorders effects on TG and APC resistance have been utilized to explore underlying mechanisms predisposing to thrombosis. Thus, in a study of patients with myeloproliferative diseases, Marchetti et al. showed an increased level of APC resistance based on TG testing, with greater resistance in JAK2(V617F) carriers compared with noncarriers, being highest in JAK2(V617F) homozygous patients. APC resistance was shown to be related to reduced protein S levels, likely the result of proteolytic cleavage by neutrophil elastase. It is tempting to suggest that these mechanisms contribute to the risk of venous (and arterial) thrombosis seen in these patients [19]. Subsequent studies confirmed the increased TG in myeloproliferative patients also in platelet rich plasma (PRP) [20], while it was also shown that part of the procoagulant activity in the TG assay resided in the microparticles fraction in plasma from the same patients [21]. This way, TG analysis can be utilized to explore underlying mechanisms of thrombosis in patients with complex cellular
H. ten Cate / Thrombosis Research 129 (2012) 367–370
disorders that involve cellular structure and function (in part determined by the acquired JAK2 (V617F) mutation), cell activation (in part reflected by microparticle formation and their functional properties) and the plasma proteome. Studies on arterial vascular disease In cardiovascular diseases associated with atherosclerosis, common genetic thrombophilic traits have less influence on the risk of acute arterial thrombosis, although the risk association is not negligible [22]. In addition, hemostatic activity may have important modulating effects on the development of atherosclerosis [23]. However, the interactions between the plasma and vessel wall compartments may be more complex than in venous thrombotic diseases. Hence, it is somewhat more difficult to predict the relationship, if any, between TG and cardiovascular disease. Atherosclerosis is typically known as an inflammatory disease [24] and the influence of inflammation perturbed vascular endothelium likely affects the plasma composition and thrombin generation reactivity. Risk factors like smoking, hypertension and external toxins like particular matter, affect thrombin generation in plasma [25]. Diseases linked to cardiovascular disease, like obesity/diabetes [26–28], chronic pulmonary disease [29] and reumatoid arthritis [30] affect thrombin formation in plasma. While, fat deposition is associated with TG [26], weight reduction in morbid obesity reduces thrombin generation [31]. In the elderly, increased TG is associated with risk of stroke, rather than myocardial infarction (MI) [32]. Also, in the elderly, risk associations between TG (F1 + 2) and cognitive functions have been exposed [33]. Some studies have now reported associations between degree of thrombin formation in plasma and (symptomatic) atherosclerotic disease, with increased TG in persons with echogenic carotid plaques [34], or with increased intima media thickness [35,36]. In a recently presented study we recognized significant associations between both TAT complexes and TG in plasma on the one hand and severity of coronary calcification on CT scan on the other hand [37]. Patients with acute MI show increased TG that persists for at least 6 months [38–43]. Since, from a diagnostic point of view, TG analysis is aimed at estimating risk of thrombosis, it is of interest to determine the predictive potential of this test. In the acute MI study a surprising observation was that although overall TG values are elevated in the acute phase as compared to controls, lower rather than higher TG levels are associated with recurrent thrombotic outcomes [40]. This is in contrast to the findings for d-dimer, which showed a more predictable positive relationship with recurrent thrombotic events in these patients. Although the origin of these paradoxical findings is not yet clear, we speculate on two possible reasons. First, these samples were taken in the acute phase of MI which may have affected the plasma composition in unpredictable ways, although analysis of clotting protein concentrations did not reveal any confounders [40]. A second thought may be that the actual lowering of TG may be due to an increased endothelial response to injury, ie by releasing tissue factor pathway inhibitor (TFPI). In a recent analysis of the RATIO case– control study in young women with MI, increased TFPI was associated with an elevated risk of MI and this was associated with an increased sensitivity of TG to APC addition [44]. These data suggest that, more than in VTE, the interaction between the arterial vessel wall (atherosclerosis) and the plasma proteome is a complex and dynamic interplay that may be in part reactive to injury and in part compensating for injury, as reflected by changes in TG. One of the obvious concerns is that these studies were mostly done in the absence of platelets and other cells that may affect TG. The next generation of whole blood TG analysis, currently under development, will provide perhaps more accurate assessments of TG in the context of arterial vascular disease. For the time being, it is
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too early to predict the clinical usefulness of TG analysis in arterial vascular diseases.
Studies on bleeding Common opinion predicts that in bleeding diatheses reduced TG would be an important contributing mechanism. Indeed, in congenital hemorrhagic diseases like the hemophilias, TG is impaired [45]. From a clinical perspective there are several arguments for investigating the potential of TG testing. First, in patients with unexplained bleeding the use of an overall assay like TG may reveal an underlying protein defect, provided the test is sufficiently sensitive to detect it (as compared to conventional clotting assays, like APTT). In a recent study of this matter, TG analysis did however not expose any differences as compared to matched controls [46]. Second, in patients with a known bleeding diathesis such as hemophilia, it is known that bleeding shows a large heterogeneity among families and individuals. Studies that addressed the potential of TG testing to characterize bleeding phenotype in terms of TG potential showed an association between TG and severity of bleeding [reviewed in 45], but whether this is clinically more relevant than measuring the level of the deficient coagulation protein remains uncertain. An interesting application may be the therapeutic monitoring of hemophilic patients that have developed inhibitors. In these cases TG analysis may be quite helpful and suggests to reflect clinical hemostasis in a series of patients undergoing surgery with replacement therapy [47]. Modification of the TG assay to measure very low levels of FVIII [48], or to monitor therapy with recombinant FVIIa [49] are interesting applications that merit further study. Another very important clinical area is peri-operative monitoring of patients at risk of bleeding. Both for diagnostic purposes and for guiding plasma component transfusion there may be a place for TG analysis in conjunction with thromboelastography. While TEG/ROTEM emerge as the principal tools for guiding peri-operative management, these methods mostly detect changes in fibrinogen levels and are less sensitive to changes in thrombin formation. A study from our group suggests that indeed peri-operative bleeding is dependent on both thrombin and fibrinogen related aspects of coagulation and that combined use of TG and TEG may be warranted for optimal transfusion management [50]. This however, needs to be further explored in prospective studies. Conclusions and Future developments After decades of research development, TG analysis by commercial methods involving fluorogenic or chromogenic substrates finally makes broad scale clinical testing possible. Standardization of test conditions is of eminent importance in order to take this assay principle towards routine clinical application. More important is to define those conditions and cutoff values (for thrombosis risk or bleeding) which would allow decision making based on individual patient data. At this stage, routine application is still in the future and prospective clinical studies will be needed to assess the diagnostic utility. Another still incompletely studied area concerns monitoring of drug therapy including low molecular weight heparins and (new) anticoagulants. Meanwhile, TG analysis provides an interesting tool for various research projects, not confined to clinical samples, but also applicable on plasma and tissue homogenates from human and animal origin. Conflict of interest statement None.
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Acknowledgements I am grateful to Prof. Coen Hemker, Prof. Jan Rosing and dr. Henri Spronk for trying to teach me the biochemistry behind thrombin generation. References [1] Spronk HM, Govers-Riemslag JW, ten Cate H. The blood coagulation system as a molecular machine. Bioessays 2003 Dec;25(12):1220–8. [2] Borissoff JI, Spronk HM, Heeneman S, ten Cate H. Is thrombin a key player in the 'coagulation-atherogenesis' maze? Cardiovasc Res 2009 Jun 1;82(3):392–403. [3] Hemker HC. Recollections on thrombin generation. J Thromb Haemost 2008 Feb;6(2):219–26. [4] Bauer KA, Rosenberg RD. The pathophysiology of the prethrombotic state in humans: insights gained from studies using markers of hemostatic system activation. Blood 1987;70(2):343–50. [5] Spronk HM, Dielis AW, Panova-Noeva M, van Oerle R, Govers-Riemslag JW, Hamulyák K, et al. Monitoring thrombin generation: is addition of corn trypsin inhibitor needed? Thromb Haemost 2009 Jun;101(6):1156–62. [6] van Veen JJ, Gatt A, Makris M. 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Contents lists available at SciVerse ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Mini Review
Thrombin generation in clinical conditions Hugo ten Cate ⁎ Dept. of Internal medicine, laboratory of Clinical Thrombosis and Haemostasis, and Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, The Netherlands
a r t i c l e
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Article history: Received 6 October 2011 Received in revised form 6 October 2011 Accepted 18 October 2011 Available online 12 November 2011 Keywords: Thrombin generation Venous thromboembolism Arterial thrombosis-atherosclerosis Bleeding
a b s t r a c t Commercial assays for determining thrombin generation in plasma are being tested in clinical conditions associated with thrombosis or bleeding. While pre-analytical conditions remain a source of inter laboratory variation, demanding for further standardization, clinical research proceeds. In patients at risk of venous thrombosis thrombin generation (TG) analysis may be utilized to detect underlying thrombophilia and this has been achieved both with addition of thrombomodulin or activated protein C, to test the contribution of the protein C system. In patients with documented venous thromboembolism, increased TG values are seen in those patients at greatest risk for recurrence, although the data are not consistent yet. In patients with arterial vascular disease, effects on TG patterns are seen that both reflect atherosclerosis (and its risk factors) and link to risk of recurrent atherothrombosis (coronary or stroke), but the data are limited. In patients with a bleeding diathesis, like hemophilia, the main importance of TG assays lies in the application for monitoring replacement therapy, either with factor concentrate or rFVIIa. An interesting application is in conjunction with thromboelastography, for monitoring peri-operative transfusion policy. Finally, TG analysis may contribute to monitoring anticoagulant drug treatment, but these and other applications would greatly benefit from whole blood, point of care applications of TG testing. © 2011 Published by Elsevier Ltd.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Methodology . . . . . . . . . . . . . . . . . . . . . . . . . Utilizing thrombin generation analysis; studies on venous thrombosis Studies on arterial vascular disease . . . . . . . . . . . . . . Studies on bleeding . . . . . . . . . . . . . . . . . . . . . . Conclusions and Future developments . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction Thrombin is a key enzyme in the blood coagulation cascade combining pro- and anticoagulant functions through controlled
Abbreviations: TG, thrombin generation; PPP, platelet poor plasma; SSC, Scientific Subcommittee of the International Society for Thrombosis and Haemostasis; VTE, venous thromboembolism; MI, myocardial infarction; APTT, activated partial trhomboplastin time. ⁎ Laboratory of Clinical Thrombosis and Haemostasis, Maastricht University Medical Center, Universiteitssingel 50, PO Box 616, 6200 MD Maastricht. Tel.: + 31 43 3884262; fax: + 31 43 3884159. E-mail address: [email protected]. 0049-3848/$ – see front matter © 2011 Published by Elsevier Ltd. doi:10.1016/j.thromres.2011.10.017
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regulation of the cascade activity [1]. Either by direct limited proteolysis of other coagulation proteins, yielding active enzymes like factor Xa, or by interacting with cellular receptors like thrombomodulin or protease activated receptors (PARs), thrombin displays multiple functions in the vasculature [2]. Because of its central position the formation of thrombin is considered one of the most important steps in coagulation. For this reason it is not surprising that investigators have tried since many years to study its formation in plasma based systems [3]. Finally, commercial methods for studying thrombin generation (TG) in plasma have now become available, allowing for an expanding number of studies on TG among the research community. In this concise review I will discuss some of the current developments in TG analysis and testing in clinical studies and try to
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delineate the perspective for using this methodology in the clinical arena. Methodology There are different approaches to the measurement of thrombin formation. An established and commercialized approach is the use of immunoassays, usually Elisa's, to quantify the activation peptide released from prothrombin upon activation by factor Xa (F1 + 2 fragment). The other Elisa approach is aimed at the thrombin antithrombin (TAT) complex. Both methods give an estimate of the amount of thrombin formed in blood at a given moment in time. Many clinical studies have employed these assays to study TG in vivo. The other approach is to quantify the amount of thrombin that can be generated ex vivo by the addition of a small amount of trigger (usually tissue factor) to a plasma sample, to assess the potential to generate thrombin, hence the name of “endogenous thrombin potential” for this type of analysis. Since both approaches are sensitive to low amounts of tissue factor and also to contact activation occurring in the course of blood sampling and processing, the quality of the plasma sample is an important preanalytical factor. For the activation peptide assays, pioneering studies by Rosenberg and Bauer on the prethrombotic state [4] considered preanalytical aspects, including the quality of the blood sample (based on degree of hemolysis etc.), but they employed radioimmunoassays. For the commercial F1 + 2 and TAT elisa's there is no good evidence suggesting specific procedures necessary other than obtaining a properly collected citrated plasma sample. This narrative review paper mainly deals with the capacity assays, in which TG is measured as thrombin potential. Several issues including the type of blood sampling device (Vacutainer® or Butterfly®), the test tube, citrate concentration and the plasma preparation have been investigated and reported [5]. One important and somewhat controversial factor is the need to prevent contact activation. While the addition of corn trypsin inhibitor (CTI) has been advocated for this purpose, CTI is expensive and only acts when already present in the test tube. In our hands, CTI was not required at tissue factor concentrations > 0.5 pmoles, however this was only tested in normal plasma, not in patients [5]. The analysis of thrombin generation is usually done with platelet poor plasma (PPP), although the method can be modified to test platelet rich plasma. The application on PPP also allows for measurement in frozen-thawed plasma from clinical studies, although there is little information about the effects of storage on the quality of the sample. When tested batchwise and including normal plasma to control for technical variation, not more than a random effect of freezing-thawing, would need to be expected. Three commercial techniques have sofar been described: the Calibrated Automated Thrombogram (Trombinoscope/Stago), the Thrombin Generation Test (Siemens) and Technothrombin TGA (Technoclone). The techniques differ considerably in terms of (concentrations of) reagents, including the use of either fluorogenic or chromogenic substrates. Although at fixed test conditions these assays may perform well within one center, comparison of these methods applied on test samples among different centers yielded major variations [6]. It therefore remains of utmost importance to clearly define test conditions, preferably using standard, reference plasma for normalization of TG values. The SSC should take the lead in this process. In the absence of such recommendations or guidelines, investigators best follow the procedures recommended by the manufacturers, at least for testing of clinical samples, in order to minimize the degree of variation imposed when homemade phospholipids, different brands of tissue factor etcetera are applied. Utilizing thrombin generation analysis; studies on venous thrombosis Analyzing the potential to form thrombin (ETP) assumes that it reflects the potential of the patient to form thrombosis, under
thrombogenic conditions like surgery or malignancy. Typically, this relationship between thrombin and thrombosis would be anticipated for venous thromboembolism (VTE), while given the lower impact of hypercoagulability in arterial thrombosis, it is a priori doubtful, whether ETP would also reflect the risk of arterial thrombosis, certainly when tested in PPP, without platelets. In subjects at risk of VTE (thrombophilia), TG analysis may add to the diagnostic workup, being able to detect intermediate thrombophilic phenotypes [6–8]. This will however require TG analysis also with the addition of thrombomodulin or activated protein C, in order to test both the basic TG and TG after full activation of the protein C system. In population based studies Lutsey et al. demonstrated an increased risk for VTE (primarily idiopathic) for elevated peak values of TG [9]. Tripodi and colleagues showed that clinical assessment of VTE risk in low, moderate or high was reflected in TG values when determined in the presence of thrombomodulin [10]. More specifically, modifications of thrombin generation analysis have been employed over the past decades to characterize both genetic and acquired thrombophilia, attributable to factor V Leiden and oral contraceptives respectively [7]. Before TG analysis can be applied in the workup of individual patients (thrombophilia screening) this will require extensive validation of test conditions, including assessment of reference values, within the routine laboratory. In patients with suspected VTE, thrombin generation analysis may help to identify low risk subjects [11], although the use of this test in the acute phase of disease may be prone to acute phase effects of depletion of clotting factors due to thrombosis that may obscure the test interpretation. Rather, in this situation, d-dimer testing may remain the first choice test for excluding thrombosis. In patients with documented VTE several studies show a predicted relationship between thrombin generation and risk of VTE. The first study by Hron et al., showed a low risk of recurrent VTE in patients with a TG value below a certain cutoff level [12]. Increased TG was associated with an increased risk of recurrent VTE in studies from Tripodi et al. [13] and Besser et al. [14], however this was not confirmed by van Hylckama Vlieg et al. finding a risk association with a first, but not with recurrent VTE events [15]. Following a first deep venous thrombosis, patients show persistently elevated TG during 24 months follow up, with reduced response to thrombomodulin, as compared to controls [16]. Whether this persistent hypercoagulability adds to single measurements of TG, to predict recurrence after cessation of anticoagulation, remains to be tested. TG analysis may be of interest in specific groups of patients at variable but elevated risk of venous thrombosis, such as those with malignancies. In the large Vienna Cancer and Thrombosis Study elevated thrombin peak values (defined as values ≥ 611 nM, being the 75th percentile) were associated with an increased risk of VTE with a hazard ratio of 2.1 (95% CI 1.3-3.3) in multivariate analysis [17]. Similarly, TG may predict increased risk of VTE in cancer patients during chemotherapy [18]. In specific malignant disorders effects on TG and APC resistance have been utilized to explore underlying mechanisms predisposing to thrombosis. Thus, in a study of patients with myeloproliferative diseases, Marchetti et al. showed an increased level of APC resistance based on TG testing, with greater resistance in JAK2(V617F) carriers compared with noncarriers, being highest in JAK2(V617F) homozygous patients. APC resistance was shown to be related to reduced protein S levels, likely the result of proteolytic cleavage by neutrophil elastase. It is tempting to suggest that these mechanisms contribute to the risk of venous (and arterial) thrombosis seen in these patients [19]. Subsequent studies confirmed the increased TG in myeloproliferative patients also in platelet rich plasma (PRP) [20], while it was also shown that part of the procoagulant activity in the TG assay resided in the microparticles fraction in plasma from the same patients [21]. This way, TG analysis can be utilized to explore underlying mechanisms of thrombosis in patients with complex cellular
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disorders that involve cellular structure and function (in part determined by the acquired JAK2 (V617F) mutation), cell activation (in part reflected by microparticle formation and their functional properties) and the plasma proteome. Studies on arterial vascular disease In cardiovascular diseases associated with atherosclerosis, common genetic thrombophilic traits have less influence on the risk of acute arterial thrombosis, although the risk association is not negligible [22]. In addition, hemostatic activity may have important modulating effects on the development of atherosclerosis [23]. However, the interactions between the plasma and vessel wall compartments may be more complex than in venous thrombotic diseases. Hence, it is somewhat more difficult to predict the relationship, if any, between TG and cardiovascular disease. Atherosclerosis is typically known as an inflammatory disease [24] and the influence of inflammation perturbed vascular endothelium likely affects the plasma composition and thrombin generation reactivity. Risk factors like smoking, hypertension and external toxins like particular matter, affect thrombin generation in plasma [25]. Diseases linked to cardiovascular disease, like obesity/diabetes [26–28], chronic pulmonary disease [29] and reumatoid arthritis [30] affect thrombin formation in plasma. While, fat deposition is associated with TG [26], weight reduction in morbid obesity reduces thrombin generation [31]. In the elderly, increased TG is associated with risk of stroke, rather than myocardial infarction (MI) [32]. Also, in the elderly, risk associations between TG (F1 + 2) and cognitive functions have been exposed [33]. Some studies have now reported associations between degree of thrombin formation in plasma and (symptomatic) atherosclerotic disease, with increased TG in persons with echogenic carotid plaques [34], or with increased intima media thickness [35,36]. In a recently presented study we recognized significant associations between both TAT complexes and TG in plasma on the one hand and severity of coronary calcification on CT scan on the other hand [37]. Patients with acute MI show increased TG that persists for at least 6 months [38–43]. Since, from a diagnostic point of view, TG analysis is aimed at estimating risk of thrombosis, it is of interest to determine the predictive potential of this test. In the acute MI study a surprising observation was that although overall TG values are elevated in the acute phase as compared to controls, lower rather than higher TG levels are associated with recurrent thrombotic outcomes [40]. This is in contrast to the findings for d-dimer, which showed a more predictable positive relationship with recurrent thrombotic events in these patients. Although the origin of these paradoxical findings is not yet clear, we speculate on two possible reasons. First, these samples were taken in the acute phase of MI which may have affected the plasma composition in unpredictable ways, although analysis of clotting protein concentrations did not reveal any confounders [40]. A second thought may be that the actual lowering of TG may be due to an increased endothelial response to injury, ie by releasing tissue factor pathway inhibitor (TFPI). In a recent analysis of the RATIO case– control study in young women with MI, increased TFPI was associated with an elevated risk of MI and this was associated with an increased sensitivity of TG to APC addition [44]. These data suggest that, more than in VTE, the interaction between the arterial vessel wall (atherosclerosis) and the plasma proteome is a complex and dynamic interplay that may be in part reactive to injury and in part compensating for injury, as reflected by changes in TG. One of the obvious concerns is that these studies were mostly done in the absence of platelets and other cells that may affect TG. The next generation of whole blood TG analysis, currently under development, will provide perhaps more accurate assessments of TG in the context of arterial vascular disease. For the time being, it is
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too early to predict the clinical usefulness of TG analysis in arterial vascular diseases.
Studies on bleeding Common opinion predicts that in bleeding diatheses reduced TG would be an important contributing mechanism. Indeed, in congenital hemorrhagic diseases like the hemophilias, TG is impaired [45]. From a clinical perspective there are several arguments for investigating the potential of TG testing. First, in patients with unexplained bleeding the use of an overall assay like TG may reveal an underlying protein defect, provided the test is sufficiently sensitive to detect it (as compared to conventional clotting assays, like APTT). In a recent study of this matter, TG analysis did however not expose any differences as compared to matched controls [46]. Second, in patients with a known bleeding diathesis such as hemophilia, it is known that bleeding shows a large heterogeneity among families and individuals. Studies that addressed the potential of TG testing to characterize bleeding phenotype in terms of TG potential showed an association between TG and severity of bleeding [reviewed in 45], but whether this is clinically more relevant than measuring the level of the deficient coagulation protein remains uncertain. An interesting application may be the therapeutic monitoring of hemophilic patients that have developed inhibitors. In these cases TG analysis may be quite helpful and suggests to reflect clinical hemostasis in a series of patients undergoing surgery with replacement therapy [47]. Modification of the TG assay to measure very low levels of FVIII [48], or to monitor therapy with recombinant FVIIa [49] are interesting applications that merit further study. Another very important clinical area is peri-operative monitoring of patients at risk of bleeding. Both for diagnostic purposes and for guiding plasma component transfusion there may be a place for TG analysis in conjunction with thromboelastography. While TEG/ROTEM emerge as the principal tools for guiding peri-operative management, these methods mostly detect changes in fibrinogen levels and are less sensitive to changes in thrombin formation. A study from our group suggests that indeed peri-operative bleeding is dependent on both thrombin and fibrinogen related aspects of coagulation and that combined use of TG and TEG may be warranted for optimal transfusion management [50]. This however, needs to be further explored in prospective studies. Conclusions and Future developments After decades of research development, TG analysis by commercial methods involving fluorogenic or chromogenic substrates finally makes broad scale clinical testing possible. Standardization of test conditions is of eminent importance in order to take this assay principle towards routine clinical application. More important is to define those conditions and cutoff values (for thrombosis risk or bleeding) which would allow decision making based on individual patient data. At this stage, routine application is still in the future and prospective clinical studies will be needed to assess the diagnostic utility. Another still incompletely studied area concerns monitoring of drug therapy including low molecular weight heparins and (new) anticoagulants. Meanwhile, TG analysis provides an interesting tool for various research projects, not confined to clinical samples, but also applicable on plasma and tissue homogenates from human and animal origin. Conflict of interest statement None.
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