Renal allograft thrombosis: can thrombophilia explain the inexplicable?

Renal allograft thrombosis is responsible for approximately 2–7% of early allograft loss [1–3]. Data from the Australian and New Zealand Dialysis and Transplant Registry demonstrate a constant rate of grafts lost due to thrombosis over the last 10 years [4]. Graft thrombosis is characterized by sudden anuria and almost inevitable irreversible loss of function. Venous thrombosis is more common, usually occurs within the first 2 weeks, and is accompanied by graft pain and swelling, frequently with allograft rupture. Arterial thrombosis may be painless and without swelling or rupture; however, thrombosis of both arterial and venous conduits can occur, and distinguishing the site of origin of thrombus may be impossible. Until recently, mechanisms for this catastrophic event were considered to reflect general and non-specific perturbations of coagulation associated with or exacerbated by the dialysis or surgical procedure, immunosuppressive drugs, technical errors, donor vessel abnormalities, vascular rejection, and recipient comorbidity (see Table 1). Whilst these factors remain as potential contributors to thrombosis, retrospective and prospective reviews have failed to demonstrate unifying or reproducible environmental risk factor(s) that could explain the often seemingly random and unexpected early graft thrombosis, making recommendations for screening and prevention impossible. Recent developments in the understanding of inherited hypercoagulable states (thrombophilia) and risk of thrombosis in the general population, have implications for understanding renal allograft thrombosis. Putative environmental risk factors for allograft thrombosis Putative environmental risk factors for allograft thrombosis Under basal conditions, a low level of blood coagulation is balanced by endogenous anticoagulant mechanisms that act to keep blood liquid and limit thrombus to sites of injury. Regulation of coagulation occurs on the endothelial surface which constitutively express an antithrombotic phenotype [5,6]. Activation of blood coagulation following trauma or inflammation alters the phenotype of the endothelium to prothrombotic with expression of subendothelial tissue factor which contacts and activates factor VII. The generation of the multifunctional clotting enzyme thrombin is ultimately the critical enzyme that converts fibrinogen to fibrin. Factors IX, X, and XI and the activated cofactors Va and VIIIa act to accelerate and amplify the conversion of prothrombin to thrombin and promote fibrin production (see Figure 1) [7]. Thrombin regulates its own action by stimulating activation of factors V and VIII, and following binding to endothelial thrombomodulin, inhibits its own activation by activating protein C. Increased levels of the peptide fragment F1+2 released by the conversion of prothrombin to thrombin are indicative of increased conversion of prothrombin to its active form [8]. Increased F1+2 in the absence of clinical thrombosis has been described in both inherited and acquired hypercoagulable states, but its measurement in clinical practice has not proven to be a reliable predictor of clinical events [9,10]. A simplified schematic representation of coagulant (green) and anticoagulant (blue) pathways occurring on the vascular endothelium. The enzyme thrombin is pro-coagulant by converting fibrinogen to fibrin, but following binding to thrombomodulin, is an anticoagulant by activating protein C (PC→APC), which cleaves activated cofactors Va and VIIIa. Generation of thrombin from prothrombin is increased (hypercoagulable) if inheritance of the mutant prothrombin 20210 A allele (red), or mutant factor Va (VaL shown in red) is present. Antithrombin III (AT III) requires endogenous heparan or exogenous heparin for its anticoagulant effects. Derived from [6,45]. The anticoagulant pathways are: (i) Tissue factor pathway inhibitor. Its role in clinical disease states is currently undefined. (ii) The protein C&S system. Protein C is a vitamin-K-dependent protein that inactivates activated cofactors Va and VIIIa. Thrombin activates protein C after binding with thrombomodulin on the endothelial surface. Activated protein C is released to combine with free protein S. Protein C is especially important for microvascular defences due to the high localization of thrombomodulin in capillaries [11]. (iii) The antithrombins (especially antithrombin III, (AT-III)). A vitamin-K-independent protease inhibitor that irreversibly neutralizes the serine proteases (factors XIIa, XIa, IXa, Xa and thrombin). Heparan sulphate on the endothelial cell, or exogenous heparin markedly enhance AT-III activity at the vascular surface interface to form a complex with the serine proteases [6]. (iv) The fibrinolytic pathway. Lysis of fibrin thrombi occurs by plasmin which is activated by endothelial tPA and inhibited by plasminogen activator inhibitors, and other antiplasmins [12]. Thrombophilia defines an inherited hypercoagulable or pre-thrombotic state [13,14]. Hypercoagulability is an increased tendency to thrombosis which may be primary (genetic) or acquired. Vessel wall injury, stasis and altered blood coagulation were suggested by Virchow to be the causes of venous thrombosis. Venous thrombosis is characterized by activation of blood coagulation in low flow states or stasis resulting in platelet poor and erythrocyte rich fibrin thrombus. Arterial thrombi are characterized by platelet-rich thrombi occurring at areas of vessel wall injury and turbulent flow. Most evidence suggests that pathological thrombi occur in genetically predisposed individuals when exposed to a clinical (environmental or acquired) stimulus for blood clotting, such as pregnancy, smoking, surgery, malignancy, or oral contraceptives [15,16]. However, primary thrombotic events unrelated to an identifiable precipitant are also characteristic of thrombophilic states. Venous thrombosis particularly affects the lower limbs, but can occur in unusual sites such as cerebral sinuses, mesenteric and renal veins, and upper limbs. Multiple changes affecting coagulation, fibrinolysis, and endogenous anticoagulant systems have been described that may contribute to thrombophilia. In addition other factors not primarily related to coagulation, such as homocysteine and antiphospholipid antibodies, are capable of contributing to thrombophilia by mechanisms that remain incompletely understood. Primary thrombophilia classically occurs with deficiency of protein C/S or AT-III (a failure of endothelial anticoagulants). However although these deficiencies are well studied and increase risk of thrombosis approximately 20-fold, they are uncommon (0.1% of the population) and account for only 6–8% of venous events [13,14]. Loss of allografts due to primary thrombophilia has been reported [17]. Secondary thrombophilia represents an acquired hypercoagulable state (cancer, oral contraceptives, pregnancy, nephrotic syndrome) and represents a state of coagulation and endothelial activation. In 1993 Dählback et al. [18] described impaired anticoagulant effect of activated protein C (APC) as a risk factor for venous thrombosis. This was shown to be due (in >90% of cases) to a specific point mutation at bp 1691 (G1691A) in the factor V molecule that results in the replacement of arginine at position 506 by glutamine (factor V Q506 or factor V Leiden). The altered cleavage site at position 506 is resistant to inactivation by activated protein C (see Figure 1) [19]. The unrestrained procoagulant function of thrombin (shown by elevated F1+2) can overwhelm the endogenous anticoagulant defences and cause pathological thrombosis. The factor V Leiden (FVL) mutation has been shown to be the most common mutation associated with venous thrombosis in European populations (approximately 5% carriers), although its allelic frequency varies according to race, being uncommon or absent in certain indigenous African and Asian populations [20]. Heterozygosity for this mutant allele increases venous thrombotic risk 5–10-fold, and homozygosity approximately 80-fold [21]. It is also a risk factor for recurrent venous thrombosis [22]. In patients presenting with their first venous thrombosis, approximately 20–60% will have APC resistance, but conversely not all patients with APC resistance suffer a clinical thrombotic event [23]. Resistance to APC does not confer an increased risk of arterial thrombosis in the general population, or increased risk of vascular access thrombosis in haemodialysis patients [24,25]. A sequence variation in the 3′ untranslated region of the prothrombin gene that is found in 2–3% of the European population, has been associated with increased prothrombin and risk of venous thrombosis [26,27]. Heterozygosity for the mutant (20210A) allele is associated with a 1.7–3-fold risk of venous but not arterial thrombosis, somewhat less than that associated with the FVL mutation [28]. Homozygosity for this mutation whilst rare has been described in individuals with and without thrombotic events. In the basal state an increased endogenous thrombin potential was noted rather than increased F1+2, suggesting that unrestrained thrombin generation in response to an appropriate stimulus rather than in vivo thrombin generation may account for its clinical phenotype [29]. Cross-linkage of fibrin by factor XIII activates fibrinolysis to degrade fibrin. Plasminogen is converted to plasmin by tissue plasminogen activator (tPA) and inhibited by plasminogen activator inhibitor-1 (PAI-1) and the antiplasmins. Whilst impaired fibrinolysis has been implicated in both arterial and venous thrombosis, measurement of specific factors and genetic polymorphisms associated with variability of proteins involved in fibrinolysis has not proved to be of particular clinical use, although an area of ongoing interest and research [12]. Impaired fibrinolysis has been linked with arterial disease through insulin resistance syndromes, hypertriglyceridaemia and raised PAI-1 [30]. Homocysteine (Hcy) is a sulphur-containing amino-acid derived from dietary methionine metabolism. Increased Hcy has been associated with both arterial [31] (in a linear relationship) and venous [32] (in a threshold effect) thrombotic risk in the general population. However, some controversy over its status as a vascular risk factor exists [33]. Increased Hcy is associated with reduced intake and lowered serum concentrations of B-group vitamins, and is invariably raised in chronic renal failure. A polymorphism (C677T) of the methylenetetrahydrofolate reductase (MTHFR) gene results in a thermolabile variant of the enzyme with reduced activity for re-methylation of Hcy. Whilst homozygosity for the mutant allele (approximately 5–15% of the population) is associated with higher Hcy [34], it remains unclear whether the risk of thrombosis is independently increased, or is limited to those with co-inherited thrombophilic states [33,35,36]. The mechanism(s) by which Hcy contributes to vascular disease is also uncertain, but endothelial toxicity along with pro-coagulant effects are described. Antibodies directed against phospholipids (APA) including cardiolipin, have been associated with both venous and arterial vascular thrombosis. These antibodies may have in vitro anticoagulant activity (lupus anticoagulant). Recent work suggests that they are largely directed against β2-glycoprotein 1 (β2GP1) and that measurement of β2GP1 is most specific for the detection of APA (reviewed in [37]). Whilst these antibodies appear to impair coagulation in vitro, in vivo they are most often associated with thrombosis. They are not confined to patients with lupus and autoimmune conditions, and can be found in some patients presenting with thrombosis, often at unusual sites [38,39]. Reports of adverse outcome following renal transplantation in patients with detectable APA are also described [40,41]. Ducloux et al. [41] detected a very high prevalence of post-transplant APA (28%), the majority of whom could be demonstrated to have had APA whilst on dialysis. There was approximately a 3-fold increase in venous (peripheral) and arterial thrombotic risk post-transplant, but only one graft was lost to thrombosis. There is a high variability in reported rates and titres of APA in dialysis patients [42,43] and continuing controversy over their significance [44], suggesting methodological aspects of their measurement and interpretation may be relevant. The relative frequency of the factor V Leiden mutation has made it possible to assess the effects of co-inheritance of thrombophilic genotypes. Co-inheritance of FV Leiden with deficiency of protein C or S, AT-III, or hyperhomocysteinaemia associated with homocystinuria significantly increase thrombotic events [45]. This suggests the importance of polygenic influence upon clinical risk. The most striking increase in risk occurs between carriers of the FV Leiden or the prothrombin G20210A mutation and the oral contraceptive when the risk of a venous thrombosis rises to 80–150-fold and thrombosis at unusual sites, such as cerebral venous sinus thrombosis can occur [46]. Why some patients develop lower-limb thrombosis and others cerebral events illustrates the complexity of gene–environmental interactions [16]. These examples suggest similar possibilities for interaction in acquired hypercoagulable states, such as renal disease/transplantation. Venous thrombosis is uncommon in patients on dialysis but is a common post-transplant complication [47]. Impaired platelet function (due in part to uraemic impairment of the platelet fibrinogen IIb/IIIa receptor with vWF) or reduced haematocrit could act as a partial protection to venous thrombosis. However, arterial thrombosis as a cause of morbidity and mortality is frequent. Chronic renal disease is associated with features of a hypercoagulable and inflammatory state: increased factor VII coagulant activity, fibrinogen, interleukin-6, and raised F1+2 consistent with increased thrombin generation [48,49]. Following renal transplantation, fibrinolysis may be impaired (at least in the long term [50]) and coagulation remains activated as a consequence of tissue trauma, inflammation and expression of tissue factor. The protein C system may be transiently depressed [51] and cyclosporin impairs the activation of protein C [52]. Coagulation and fibrinolysis within the transplanted kidney will be balanced on the donor kidney endothelium, which may be subject to trauma from reperfusion injury, activation of a pro-coagulant surface by cytokines and suffer the effects of recipient immune response [5]. If the endothelium is therefore primed to exhibit a prothrombotic state, and the recipient's defences are impaired by genetic and/or acquired predisposition to a hypercoagulable state, conditions for thrombosis may be met. This hypercoagulable state following transplantation appears to persist throughout life [53], but several studies suggest venous risk is maximal in the first 6 months [47,54]. Specific allograft thrombosis is most often an early event, suggesting immediate overwhelming of local defences by an exuberant pro-coagulant state. Over-expression of PAI-1 by donor endothelium for example has been demonstrated in grafts lost to venous thrombosis, suggesting impaired regional fibrinolysis [55]. Variation in recipient PAI-1 genotype was associated with a modest 2-fold increase in thrombotic risk for carriers of the 4G allele (with increased PAI-1 activity) compared with the 5G allele (who have reduced PAI-1 activity) following renal transplantation, consistent with a genetic contribution to thrombosis [56]. Whilst allograft thrombosis occurred in the pre-cyclosporin era, the introduction of this agent led to reports of both in vivo and in vitro thrombogenicity along with its increased efficacy [1,47,57–60]. Since allograft rejection was more frequent and vigorous pre-cyclosporin, and graft thrombosis often attributed to rejection, it is difficult to review rates of thrombosis between the cyclosporin and non-cyclosporin eras retrospectively. Undoubtedly cyclosporin has multiple in vitro procoagulant effects, including activation of monocytes to express tissue factor [61], increased platelet aggregation [62], endothelial dysfunction and activation of the intrinsic coagulation pathway [63,64], impaired fibrinolysis [65], and impaired activation of protein C [52]. Whilst some studies [57,60,66] have suggested clinical hypercoagulability, the largest retrospective [2] or prospective [67] trials examining this have failed to support a significant difference in thrombosis events between cyclosporin-treated patients and non-cyclosporin-treated patients. Since no study controlled for the possibility of thrombophilia, variability in these studies may well reflect confounding by inherited hypercoagulability. Cyclosporin remains a cornerstone of contemporary transplantation, making it difficult to resolve these issues fully, but its continuing widespread use implies that clinicians consider its benefits to outweigh any potential thrombotic risk. Recently two retrospective studies have confirmed the potential relevance of inherited thrombophilia and renal allograft thrombosis. In 1997 Irish et al. [68] reported a 6% prevalence of FV Leiden in 300 transplant recipients. Carriers of the FVL mutation had a 4-fold increase in allograft thrombosis, accounting for 20% of primary allograft loss. This was the first study to confirm both the risk of this mutation (probably an underestimate due to confounding), but also confirm the heterogeneity of its expression as the majority of carriers did not experience a thrombotic event. There was no increased risk of arterial thrombosis. In addition there was a higher percentage of family history of thrombosis in patients who experienced a thrombotic event, compared with those free of thrombosis, consistent with an inherited risk of thrombosis due to other (unidentified) factors. Successful retransplantation with heparin prophylaxis was possible in two patients. In 1998 Fischereder et al. [69] described a prevalence of thrombophilia of 14% (FV Leiden 8%, LA 5%, protein S deficiency 1%) in patients. They reported a increase risk of graft loss at 1 although it is unclear whether this thrombotic or graft suggesting a more chronic of grafts from thrombophilia. these studies are consistent with thrombophilia contributing to risk of allograft thrombosis. study has the prothrombin G20210A mutation and thrombotic although it is to that it is to be associated with an adverse graft all patients a kidney transplant be for thrombosis, although is The that allograft thrombosis is not a random event, but a complex of genetic and acquired risk factors of screening to risk and The of this by all is if and and of a risk the use of an to that risk. However, to allograft thrombosis have not been in controlled for prophylaxis of venous thrombosis have been on risk although no specific for is Renal transplant for venous thrombosis are as high risk of and in patients recommendations and In the absence of specific several are all transplant at high risk and or to risk and prophylaxis whilst transplant will have to consider for its several can be a clinical of the clinical risk factors be considered (see Table with no clinical risk screening for APC resistance and the prothrombin gene mutation is of their high frequency in the general and their risk status for venous thrombosis. of these also for thrombotic risk and risk of risk of interaction with oral and pregnancy, for increased clinical and for family in of clinical risk transplant In patients at higher risk a thrombophilia protein S and be The heterogeneity of antiphospholipid and their interpretation it more difficult to β2GP1 or a lupus anticoagulant be most The by Fischereder et al. [69] and Ducloux et al. [41] suggest that and post-transplant APA have a higher risk of poor graft early prophylaxis with heparin or with is in this is not proven as a risk factor for allograft thrombosis, the invariably high Hcy levels in renal disease and its interaction with FVL suggest it as a for with vitamins, particularly in of some evidence for its with arterial vascular disease in this The role of the genotype and thrombotic risk remains and it appears to screening for this and of patients at high risk of allograft thrombosis and of patients at high risk of allograft thrombosis to risk of lower thrombosis are for all patients and and early studies have or heparin for prophylaxis of rejection or allograft thrombosis in or transplant recipients. In at high in a significant in vascular thrombosis when compared with a et al. transplant patients to heparin for or no thrombotic events occurred in the heparin compared with events in patients in the although this was not Most kidney prophylaxis with for the of or for at least 1 risk was as a hypercoagulable state or multiple There were no allograft and no events. there was no this study is the first to both a screening and a prophylaxis with according to risk. The results but are that is possible with support early of to patients. a can the efficacy and of with heparin or other the and of more specific and for the coagulation such as the antithrombin and its the increased thrombin associated with prothrombin gene mutation for will be for use in risk and in renal Renal allograft thrombosis remains a cause of early allograft thrombosis. It not be considered an and consequence of from the general and early from renal patients the that the of of renal disease with inherited thrombophilia, and the altered environmental of transplantation to thrombosis in Figure not the of a constant of grafts to thrombosis and to to risk factors and confirm appropriate screening and for its almost In the as the of gene with for thrombophilia in patients at risk will to and thrombosis with from the of The contribution of thrombophilia to mechanisms of chronic allograft loss is also a important but area of A schematic representation of the interaction between acquired of renal disease states, inherited genetic that contribute to risk of thrombosis, and the of an environmental such as surgery, in clinical events. The of inherited genetic for thrombosis may be the critical factor in risk of a clinical event. from