Department of Nephrology, Royal Perth Hospital, Perth, Western Australia, Australia
Correspondence and offprint requests to: Ashley Irish FRACP, Department of Nephrology, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6001.
Introduction
Renal allograft thrombosis is responsible for approximately 27% of early allograft loss [13]. 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.
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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].
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Thrombophilia
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 68% 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.
Activated protein C resistance
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 510-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 2060% 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].
Prothrombin gene G20210A polymorphism
A sequence variation in the 3' untranslated region of the prothrombin gene that is found in 23% 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 1.73-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].
Fibrinolysis
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
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 515% 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.
Antiphospholipid antibodies and the lupus anticoagulant
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 ß2-glycoprotein 1 (ß2GP1) and that measurement of ß2GP1 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.
Genegene and geneenvironment interactions
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 80150-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 geneenvironmental interactions [16]. These examples suggest similar possibilities for interaction in acquired hypercoagulable states, such as renal disease/transplantation.
Renal disease and 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].
Cyclosporin and allograft thrombosis
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 [1,47,5760]. 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.
Primary thrombophilia and allograft thrombosis
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 132 patients. They reported a 3.5-fold increase risk of graft loss at 1 year, although it is unclear whether this represented thrombotic or combined thrombotic/rejection graft loss, suggesting a more chronic attrition of grafts from thrombophilia. Although retrospective, these studies are consistent with thrombophilia (particularly FVL) contributing to risk of allograft thrombosis. No study has examined the prothrombin G20210A mutation and thrombotic risk, although it is reasonable to assume that it is likely to be associated with an adverse graft outcome.
Screening
Should all patients receiving a kidney transplant be screened for thrombophilia? Allograft thrombosis, although uncommon, is potentially preventable. The recognition that allograft thrombosis is not simply a random event, but probably reflects a complex interplay of genetic and acquired risk factors allows consideration of screening to stratify risk and allow rational intervention. The increasing shortage of organ donors demands preserving this valuable resource by all means possible. Screening is worthwhile if simple and inexpensive, and identification of a recognized risk allows the use of an effective intervention to reduce that risk. However, measures to reduce allograft thrombosis have not been validated in controlled randomized trials. Recommendations for prophylaxis of venous thrombosis have been published [70,71] based on risk stratification, although no specific advice for organ recipients is given. Renal transplant risks for venous thrombosis are probably classified as high (48% risk of proximal-vein thrombosis), and in non-renal patients treatment recommendations include unfractionated heparin, low-molecular-weight heparin, and intermittent pneumatic calf compression [71]. In the absence of specific trials, several options are available. Continue current practice, treat all transplant recipients at high risk and initiate universal prophylaxis, or attempt to stratify risk and modify prophylaxis accordingly. At present, whilst each transplant unit will have to consider for itself its policy, several points can be suggested. First, a careful clinical assessment of the recipients clinical risk factors should be considered (see Table 2). For people with no clinical risk factor, screening for APC resistance and the prothrombin gene mutation is recommended because of their high frequency in the general population and their established risk status for venous thrombosis. Identification of these mutations also gives information for life-long thrombotic risk and risk of recurrence, risk of interaction with oral contraception and pregnancy, allows for increased clinical surveillance, and provides information for family members in advance of clinical risk (e.g. elective transplant donation) [23,72]. In patients at higher risk a full thrombophilia screen (including protein C, S and AT-III) should be considered. The heterogeneity of antiphospholipid tests and their interpretation makes it more difficult to recommend universal screening. ß2GP1 or a functional lupus anticoagulant test would be most specific. The data by Fischereder et al. [69] and Ducloux et al. [41] suggest that (pre-) and post-transplant APA have a higher risk of poor graft outcome. Whether early prophylaxis with heparin or long-term anticoagulation with warfarin is indicated in this circumstance is unknown, however. Although not proven as a risk factor for allograft thrombosis, the invariably high Hcy levels seen in renal disease and its interaction with FVL carriers, suggest it as a candidate for treatment with high-dose B group vitamins, particularly in view of some evidence for its association with arterial vascular disease in this group. The role of the MTHFR genotype and thrombotic risk remains unclear, and it appears premature to recommend screening for this mutation.
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General measures to reduce risk of lower limb thrombosis are recommended for all patients and include calf-stimulators, graduated compression stockings, and early mobilization [70]. Four studies have used unfractionated [73,74] (n=2) or low-molecular-weight heparin [75,76] (LMWH) (n=2) for prophylaxis of rejection [73] (n=1) or allograft thrombosis (n=3) in adults [74,76] or paediatric [75] transplant recipients. In children selected at high risk, enoxaparin resulted in a significant reduction in vascular thrombosis when compared with a historic control group [75]. Ubhi et al. [74] randomized 70 consecutive transplant patients to either 5000 U subcutaneous heparin twice daily for 7 days, or no heparin. No thrombotic events occurred in the heparin group compared with six events in five patients in the heparin-free group, although this was not statistically significant. Most recently 120 adult kidney recipients received prophylaxis with dalteparin 2500 Units daily (low-risk group) for the period of hospitalization only, or 5000 U daily (high-risk) for at least 1 month [76]. High risk was defined as a hypercoagulable state (15%) or multiple vessels (31%). There were no allograft thromboses and no major haemorrhagic events. Although there was no control group, this study is the first to adopt both a screening programme, and a structured prophylaxis with LMWH according to risk. The results require confirmation but are encouraging that preventive treatment is possible with low-risk, and, pending further studies, support early administration of LMWH to high-risk patients. Only a large multi-centre randomized trial can determine the safety, efficacy and optimal duration of therapy with heparin or other agents. Additionally, the development and availability of more specific inhibitors and antagonists for the coagulation pathway, such as the antithrombin hirudin and its derivatives [77] (for the increased thrombin associated with prothrombin gene mutation for example) will be available for use in defined risk situations and await evaluation in renal transplantation.
Summary
Renal allograft thrombosis remains a preventable cause of early allograft thrombosis. It should not be considered simply an unpredictable and poorly understood consequence of surgery. Extrapolated data from the general population and early data from renal patients supports the concept that the interplay of non-inherited hypercoagulability of renal disease with inherited thrombophilia, and the altered environmental milieu of transplantation predisposes to thrombosis (summarized in Figure 2). We should not accept the inevitability of a constant attrition of grafts to thrombosis and need to continue to identify risk factors and confirm appropriate screening and interventions for its prevention, almost certainly requiring collaborative multi-centre trials. In the future, just as we now expand the specificity of HLA gene typing with molecular biology, genotyping for recognized thrombophilia genes in patients at risk will expand our ability to recognize and prevent thrombosis with targeted interventions drawn from the increasing array of anticoagulants now available. The contribution of thrombophilia to non-immune mechanisms of chronic allograft loss is also a potentially important but neglected area of research.
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References