The Pl A2 Polymorphism of the Platelet Glycoprotein IIIA Gene as a Risk Factor for Acute Renal Allograft Rejection

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1 J Am Soc Nephrol 10: , 1999 The Pl A2 Polymorphism of the Platelet Glycoprotein IIIA Gene as a Risk Factor for Acute Renal Allograft Rejection EDUARDO SALIDO,* BASILIO MARTÍN,* YSAMAR BARRIOS,* JOSÉ D. LINARES,* DOMINGO HERNÁNDEZ,* MARIAN COBOS,* MARÍA D. CHECA, LUIS HORTAL, ANA FERNÁNDEZ, JOSÉ J. GARCÍA, and ARMANDO TORRES* *Servicio de Nefrología and Unidad de Investigación, Hospital Universitario de Canarias, Hospital Insular de Las Palmas, Hospital Nuestra Señora del Pino; and Hospital Ntra. Sra. de la Candelaria, Canary Islands, Spain. Abstract. Glycoprotein IIIa/IIb is a membrane receptor for fibrinogen and von Willebrand factor that plays an important role in platelet aggregation. The integrin chain of this receptor, GPIIIa, is polymorphic, and the allele known as Pl A2 has been associated with coronary thrombosis. The GPIIIa genotype of a cohort of 119 consecutive renal allograft recipients ( yr; 85 M/34 F; 24.4% diabetic patients) was determined by PCR-restriction fragment length polymorphism, and those patients were followed for at least 12 mo. From 119 patients with at least 1 yr of follow-up, those who suffered an acute rejection (n 52) showed a lower proportion of HLA-DR 1 identity with the donor (7.7% versus 23.9%; P 0.03), a higher proportion of cytomegalovirus-positive (CMV ) donors/cmv recipients (21% versus 7.5%; P 0.05), and the Pl A2 allele was more frequent (48.1% versus 26.9%; P 0.02) compared with patients free of acute rejection (n 67). No other variable was associated with acute rejection in the univariate analysis. The impact of the three above-mentioned significant variables on acute rejection was analyzed by stepwise logistic regression. The presence of the Pl A2 allele yielded an odds ratio of 2.75 (95% confidence interval, 1.01 to 7.93) and an HLA-DR 1 identity of 0.2 (95% confidence interval, 0.06 to 0.99) for suffering an acute rejection episode. In addition, the serum creatinine at discharge was higher in Pl A2 -positive versus Pl A2 -negative patients ( versus mg/dl, respectively; P 0.01), and the prevalence of proteinuria 1.5 g/d 1 yr after transplantation was significantly higher among patients showing the Pl A2 allele (16% versus 3%; P 0.02). Finally, in the entire cohort of patients, the 2-yr graft survival was significantly lower in Pl A2 -postitive (n 43) compared with Pl A2 -negative (n 76) patients (85.7% versus 97.2%; P 0.015). No differences were found in patient survival (95.2% versus 98.7%, respectively). Proportional hazards regression analysis (Cox regression model) confirmed that serum creatinine level at discharge is the best predictor of allograft survival, followed by CMV status, delayed graft function, and the glycoprotein IIIa/IIb genotype. The Pl A2 polymorphism is an independent risk factor for acute renal graft rejection, affecting short-term graft survival. Future studies aimed at preventing the hemostatic imbalance favoring platelet aggregation associated with this polymorphism may be important in preventing acute rejection and its impact on chronic rejection. Platelets play an important role in renal allograft rejection. The allograft vascular endothelium is damaged not only as a consequence of the host immune response, but also by pharmacologic, mechanic, and hemodynamic factors. The accumulation of platelets within the rejecting allografts, usually ascertained by 111-In-labeled platelet scintigraphy, has long been reported as evidence of their importance in vascular rejection (1). In addition, direct morphologic evidence suggests that platelet aggregation on the allograft endothelial cells is an adverse Received March 16, Accepted June 16, Correspondence to Dr. Armando Torres, Unidad de Investigación, Hospital Universitario de Canarias, Ofra-La Laguna, E Tenerife, Spain. Phone: ; Fax: ; atorres@huc.rcanaria.es and esalido@ull.es / Journal of the American Society of Nephrology Copyright 1999 by the American Society of Nephrology prognostic sign during the course of rejection (2). After platelets adhere to the subendothelial matrix, the clotting cascade is activated, resulting in fibrin deposition. Endothelial, inflammatory, and platelet cells then release thromboxanes, leukotrienes, platelet activating factor, and platelet-derived growth factor, which are crucial mediators of both acute and chronic allograft vascular lesions (reviewed in reference (3). The platelet glycoprotein IIb/IIIa receptor (GP IIb/IIIa) is the most abundant integrin on the platelet surface, and it has a central role in platelet aggregation, although it is also involved in platelet adhesion, the initial step in homeostasis (4). GP IIb/IIIa consists of a 136-kD subunit and a 92-kD chain, resulting in heterodimers that bind fibrinogen, von Willebrand factor, fibronectin, vitronectin, and a number of other adhesive proteins (reviewed in reference (5). Binding of fibrinogen to GP IIb/IIIa is the principal mechanism for platelet aggregation, while interaction with von Willebrand factor seems to be critical for platelet aggregation under high shear conditions (6).

2 2600 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 GP IIb/IIIa, like other glycoproteins of the platelet membrane, is highly polymorphic, and the alloantigen known as Pl A is the most frequently involved in immune-mediated platelet destruction. Pl A1 -negative individuals are known to produce anti-pl A1 antibodies that react with platelet GPIIIa (7). The molecular basis of this polymorphism is a T-to-C change at position 1565 in exon 2 of the GP IIIa gene (also known as integrin 3- ITGB3 gene). As a result, Pl A1 molecules have a leucine, whereas Pl A2 proteins have a proline, at position 33 of the mature 3 integrin chain (8). The Leu33Pro amino acid substitution is likely to result in significant conformational changes in the structure of the GPIIb/IIIa receptor, as inferred from the nature of the amino acids involved and also from the notable immune reaction elicited by the Leu-33-GPIIIa protein (Pl A1 variant) among homozygous Pl A2 individuals. However, direct experimental evidence is lacking about the consequences of the Leu33Pro change in ligand binding to GPIIb/IIIa and platelet aggregation in general. Nevertheless, recent studies have shown a strong association between the Pl A2 polymorphism of the GPIIIa gene and acute coronary thrombosis (9), which could be interpreted as indirect evidence that the Pro33 form of GPIIIa is somehow associated with a hemostatic imbalance favoring platelet aggregation. In fact, preliminary evidence seems to indicate that the Pl A2 polymorphism conveys increased platelet aggregability (10). In the present study, we have analyzed the influence of the Pl A2 polymorphism of the GPIIb/IIIa receptor on kidney allograft rejection and survival in 119 consecutive patients. Materials and Methods Patients From February 1996 to October 1997, we prospectively studied a cohort of 122 consecutive recipients of a cadaver kidney graft transplanted at the University Hospital of Tenerife, the reference Center of the Canary Islands Renal Transplant Program. We excluded three early graft losses due to technical failure. All remaining 119 patients completed a minimum follow-up of 1 yr. Recipient and donor demographics are listed in Table 1. The kidneys were procured according to standard techniques from Table 1. Recipient (n 119) and donor demographics Recipient age (yr) Recipient gender (M/F) 85/34 Diabetic patients (%) 29 (24.4%) Time on dialysis (mo) Panel reactive antibodies 25% 14 (11.8%) Transfusions HLA ABDR mismatches HLA-DR 1 mismatches Retransplant (%) 9 (7.6%) Donor age (yr) Donor gender (M/F) 84/35 Cold ischemia time (h) Revascularization time (min) Delayed graft function (%) 28 of 119 (23.5%) the four reference hospitals of the Canary Islands. The preservation solution for cold storage on ice consisted of University of Wisconsin Solution as part of a multiorganic harvesting in 72% of the cases. In the remaining 28%, a flush solution containing mannitol was used (M-400, Barcelona) (11). Immunosuppression Immunosuppression consisted of a 7-d course of antithymocyte globulin (ATGAM; Upjohn, Kalamazoo, MI) for induction, and prednisone, cyclosporine, and azathioprine for maintenance (12). Recipients older than 50 yr did not receive azathioprine. Induction with OKT3 (5 mg/d) (Ortho Pharmaceutical, Raritan, NJ) was performed in patients at immunological risk; in addition, 13 of them received mycophenolate mofetil (2 g/d) instead of azathioprine, as a part of the triple immunosuppression. The dose of prednisone was 0.3 mg/kg body wt per d during the first 3 mo, and then it was gradually reduced to 10 mg/d by 1 yr. Cyclosporine was started when plasma creatinine was 3 mg/dl, at 8 mg/kg body wt per d, and then adjusted according to the total blood levels. The dose of azathioprine was 1.25 to 1.50 mg/kg body wt. Episodes of acute rejection were initially treated with three boluses of 500 mg of intravenous methylprednisolone. Resistant episodes or episodes with a Banff score IIb (13) were treated with a 10-d course of OKT3 (5 mg/d) (Ortho Pharmaceutical). Graft Monitoring Postoperative imaging studies (renal scintigram and color Doppler ultrasound) were obtained if the urine output was 50 ml/h during the early postransplant period or if the creatinine failed to decrease appropriately. Delayed graft function was defined as the need for at least one dialysis session during the first 7 postoperative days. Renal allograft dysfunction was assessed by clinical and laboratory methods, including imaging techniques and transplant aspiration cytology. Cyclosporin levels were monitored and nonimmune causes of allograft dysfunction were first ruled out. Then, a positive diagnosis of acute allograft rejection was reached based on serum creatinine levels, plus either renal scintigram or Doppler ultrasound, and fine needle aspiration cytology indicative of rejection (corrected increment value 3) as described previously (14). When necessary, core biopsy was used to secure the diagnosis (30% of rejection episodes) and rejections were classified according to the Banff criteria (13). Genomic Typing of the GPIIIa (ITGB3) Leu33Pro Polymorphism The previously described Pl A1 (Leu33) and Pl A2 (Pro33) alleles of the GP IIIa gene (7) were ascertained by PCR amplification of a fragment of intron 1 and exon 2 of the GP IIIa gene (15), followed by restriction enzyme digestion with MspI. DNA was purified from 3 ml of blood by proteinase K digestion, phenol extraction, and ethanol precipitation, following standard protocols. Approximately 0.1 g of DNA was amplified with primers GP3 1: 5 -CTTAGCTATTGG- GAAGTGGTAGG-3 and GP3 2: 5 -ACTGACTTGAGTGACCT- GGGAG-3, which yield PCR products of 256 bp. PCR conditions included 10 mm Tris-HCl, ph 9.0, 50 mm KCl, 1.5 mm MgCl 2, 0.1% Triton X-100, 0.1 mm dntp, 0.5 M of each primer, and 1 U Taq DNA polymerase. A Perkin-Elmer 480 thermal cycler was used to carry out 30 amplification cycles with the following temperature profile: 94 C (1 min), 62 C (1 min), and 72 C (1 min). Seven microliters of the amplification product was digested with 10 U MspI (Promega) at 37 C for 3 h, resolved by electrophoresis in 5% polyacrylamide gels, and visualized under ultraviolet light after staining with 0.5 g/ml ethidium bromide. The Pl A1 allele (Leu33) remains as

3 J Am Soc Nephrol 10: , 1999 Pl A2 Polymorphism in Renal Transplantation 2601 a 256-bp band, while the Pl A2 allele (Pro33) yields two bands of 154 and 102 bp after MspI digestion. These conditions consistently resulted in robust digestions, and both positive and negative control samples were included in the analysis to detect potential partial digestions. Genotype results were entered in the database and analyzed at the end of the study, and this information was not available to the nephrologist at the time of diagnosis of allograft dysfunction. One hundred (50 males and 50 females) randomly chosen samples from our blood bank were used to ascertain the GPIIIa allelic frequencies in our population. Statistical Analyses All categorical variables were analyzed using 2 test or Fisher exact probability test (two-tailed) as appropriate. For univariate comparison of numerical variables, t test was used. The results of the univariate analysis were considered, without correcting for the number of variables tested, only as indication of variables that deserved further investigation. The general factorial ANOVA was used when the effect of two independent variables (factors) on a dependent numerical variable was examined, while adjusting for a confounding covariate. We determined the impact on the incidence of acute rejection of those variables considered as significant risk factors in the univariate analysis by means of forward stepwise logistic regression analysis. Graft and patient survival curves were calculated using the Kaplan Meier method; survival rates between different groups were compared using the log-rank test. Proportional hazards regression of independent variables was performed with Cox regression model. All data are expressed as mean SD, and a P value 0.05 was considered significant. All computations were made using the Statistical Package for the Social Sciences, version 7.5 for Windows (Chicago, IL). Results A representative sample of our genotyping assay is shown in Figure 1, which includes the three possible Pl A allelic combinations. The Pl A2 allele frequency among the 100 blood bank samples was 18% (66 individuals were homozygous for Pl A1, 2 were homozygous for Pl A2, and 32 were heterozygous Pl A1/ A2). These figures are similar to those found among the renal allograft recipients (Pl A2 allele frequency 19%), and the percentages of the various genotypes were not different from expected values from the Hardy Weinberg postulate. Table 2 shows the demographic and clinical data of those recipients with a minimum follow-up of 12 mo, classified by the presence (Pl A2 ) or absence (Pl A2 ) of the Pl A2 allele. The proportion of patients with an acute rejection episode as well as the number of episodes per patient were significantly higher in Pl A2 patients. In addition, plasma creatinine levels at discharge were higher in the Pl A2 group. However, a trend toward higher donor age and a higher prevalence of delayed graft function was observed in this group. Thus, a general factorial analysis was applied using the number of acute rejection episodes as the dependent variable, and both the Pl A2 and delayed graft function status as factors, while adjusting for the donor age. The mean adjusted episodes of acute rejection were again significantly higher in Pl A2 patients ( versus , respectively; P 0.046). In addition, no effect of delayed graft function on the number of rejection episodes ( in early function versus in delayed graft function) Figure 1. Analysis of the Pl A2 polymorphism by PCR-restriction fragment length polymorphism. DNA amplification with GPIIIa-specific primers spanning the exon 2 polymorphic sequence results in a 256-bp band (lane 8). MspI digestion of the PCR products yields either an MspI-resistant, 256-bp band (Pl A1 allele) or two bands of 154 and 102 bp (Pl A2 allele). Samples 1, 4, 6, and 7 correspond to homozygous Pl A1/A1 individuals, whereas lane numbers 2 and 3 are heterozygous Pl A1/A2, and lane 5 corresponds to one of the two homozygous Pl A2/A2 included in our study. Lane M is the molecular weight standard. Digestion products have been separated in 5% acrylamide gel electrophoresis and stained with ethidium bromide. or an interaction of Pl A2 and delayed function was observed (P 0.34). The same analysis was performed using the serum creatinine at discharge as the dependent variable. The adjusted serum creatinine at discharge was higher in Pl A2 than in Pl A2 patients ( versus mg/dl, respectively; P 0.02), as well as in patients with delayed graft function versus those with early function (2.8 2 versus mg/dl, respectively; P ). Interestingly, an interaction of the Pl A2 polymorphism and the delayed graft function status on serum creatinine at discharge was found (P 0.04). Finally, the prevalence of proteinuria 1.5 g/d 1 yr after transplantation was more common among patients showing the Pl A2 allele (16% versus 3%; P 0.02); these patients also showed a trend toward higher serum creatinine 1 yr after transplantation. As a consequence of the increased incidence of acute rejection, the cumulative dose of methylprednisolone was higher among the Pl A2 patients ( versus ; P 0.04). In addition, a somehow higher frequency of OKT3 rescue therapy in the Pl A2 group (6 of 25 versus 3of27;P 0.28) reflected a higher incidence of vascular rejection episodes in this group (see below). All biopsies were scored according to the Banff criteria (13), and the pathologist did not have the patients genotype information available during this process. Among the 10 Pl A2 patients who underwent core biopsy, six showed vascular lesions (endothelialitis, endarteritis, etc.), which qualified them as either type IIb (n 4) or type III (n 2) rejection. Similar lesions (type IIb acute rejection) were present in only one of the eight Pl A2 patients who underwent core biopsy (P 0.065). Forty-two percent of the patients (35% of Pl A2 and 45% of the Pl A2 patients; P 0.45) received antiplatelet medication

4 2602 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Table 2. Demographic and clinical data of 119 patients with a minimum follow-up of 12 mo, classified by the presence (PI A2 ) or absence (PI A2 ) of the PI A2 allele a Characteristic PI A2 (n 43) PI A2 (n 76) P Value Receptor demographics age (yr) gender (M/F) 35/8 51/ diabetic (%) 7 (16.3%) 22 (28.9%) 0.18 Donor demographics donor age donor cardiovascular death 20 (46.5%) 40 (52.6%) 0.57 donor creatinine (mg/dl) Ischemia/delayed function cold ischemia (h) revascularization time (h) delayed graft function 14 (32.6%) 14 (18.4%) 0.11 Immunologic status HLA ABDR mismatches genomic HLA-DR 1 mismatches panel reactive antibodies 25% 3 (7.0%) 11 (14.5%) 0.37 transfusions retransplant 2 (4.6%) 7 (9.2%) 0.49 Immunosuppression OKT3 induction 14 (32.5%) 27 (35.5%) 0.84 OKT3 rescue therapy 6 of 25 (24.0%) 3 of 27 (11.1%) 0.28 mycophenolate treatment 7 (16.3%) 6 (7.9%) 0.22 cumulative MP dose (g) CsA levels at discharge (ng/ml) CsA levels at 12 mo (ng/ml) CMV infection CMV status (donor /recipient ) 9 (20.9%) 12 (15.8%) 0.62 CMV disease 11 (25.6%) 11 (14.5%) 0.15 Acute rejection acute rejection episodes patients with acute rejection 25 (58.1%) 27 (35.5%) 0.02 late acute rejection ( 6 mo) 6 of 25 (24.0%) 8 of 27 (29.6%) 0.76 biopsy with vascular lesions 6 of 10 (60.0%) 1 of 8 (12.5%) Renal function creatinine at discharge (mg/dl) creatinine at 12 mo (mg/dl) proteinuria at 12 mo (g/d) proteinuria 1.5 g/d at 12 mo 6 of 38 (15.8%) 2 of 74 (2.7%) 0.02 episodes of acute CsA toxicity a CsA, cyclosporin A; CMV, cytomegalovirus. (aspirin in 75% of the cases and ticlopidine in the remaining 25%) at any time during the course of the study, as clinically indicated by the consultant nephrologist of each Center. The number of acute rejection episodes per patient as well as the percentage of patients suffering an acute rejection episode were very similar regardless of whether the patients received an antiplatelet drug ( versus and 45% versus 36%, respectively). A univariate analysis of the factors potentially associated with the occurrence of acute rejection was performed (Table 3). Of the many variables analyzed, cytomegalovirus (CMV) status (donor /recipient ), HLA-DR 1 identity, and the GPIIIa polymorphism showed an association, while delayed graft function showed a trend. Thus, a forward stepwise logistic regression analysis was performed using the appearance of an acute rejection episode as the dependent variable, and the four above-mentioned variables as the predictors. HLA-DR 1 identity (odds ratio 0.2 [95% confidence interval, 0.06 to 0.99]; P 0.03) and the presence of the Pl A2 allele (odds ratio 2.75 [95% confidence interval, 1.01 to 7.93]; P 0.04) were the

5 J Am Soc Nephrol 10: , 1999 Pl A2 Polymorphism in Renal Transplantation 2603 Table 3. Analysis of the variables associated with acute rejection in 119 recipients with a minimum follow-up of 1 yr after transplantation Variable No Rejection (n 67) Acute Rejection (n 52) P Value Recipient age (yr) Donor age (yr) HLA ABDR incompatibilities HLA-DR 1 identity (genomic) (%) 16 (23.9%) 4 (7.7%) 0.03 Presence of PI A2 allele (%) 18 (26.9%) 25 (48.1%) 0.02 Panel reactive antibodies 25% 6 (9.0%) 8 (15.4%) 0.39 Transfusions Retransplant (%) 5 (7.5%) 4 (7.7%) 1 Cold ischemia (h) Revascularization time (min) Delayed graft function (%) 12 (17.9%) 16 (30.8%) 0.13 Induction with OKT3 (%) 26 (38.8%) 15 (28.8%) 0.33 CMV status (donor /recipient ) 5 (7.5%) 11 (21.2%) CsA levels at discharge (ng/ml) CsA levels 12 mo (ng/ml) significant predictors. All relevant factors left out of the final stepwise regression model were added in, one at the time, to recheck that they were not important predictors. Donor age (P 0.65), receptor age (P 0.26), serologic HLA mismatch (P 0.95), panel reactive antibodies 25% (P 0.29), transfusions (P 0.47), retransplant (P 0.24), cold ischemia time (P 0.10), revascularization time (P 0.78), delayed graft function (P 0.10), and CMV disease (P 0.20) all failed to modify the effect of the Pl A2 polymorphism on the prediction of rejection. In the entire cohort of patients, the 2-yr graft survival was significantly lower in Pl A2 (n 43) compared with Pl A2 (n 76) patients (85.71% versus 97.22%; P 0.015) (Figure 2). Proportional hazards regression analysis (Cox regression model) was performed introducing as independent variables the glycoprotein IIIa/IIb genotype and all variables known to influence allograft survival (presence of rejection, delayed graft function, donor age, HLA-DR 1 identity, CMV status, and serum creatinine levels at discharge). The results (Table 4) confirm that serum creatinine level at discharge is the best predictor of allograft survival, followed by CMV status, delayed graft function, and the glycoprotein IIIa/IIb genotype. Patient survival was not different between Pl A2 and Pl A2 patients (95.2% versus 98.7%, respectively). Discussion Our data demonstrate an association between the Pl A2 polymorphism of glycoprotein IIIa and the occurrence of renal Table 4. Cox regression model (proportional hazards regression) to analyze the risk factors for allograft survival a Variable Relative Risk (95% CI) P Value Figure 2. Graft survival of 119 renal transplant patients as a function of the presence (n 43) or absence (n 76) of the Pl A2 allele of the GPIIIa gene. Patients with this allele showed a significantly lower graft survival. Creatinine at discharge 2.44 (1.51 to 4) CMV status (donor / 2.1 (1.14 to 3.76) 0.02 recipient ) Delayed graft function 1.96 (1.05 to 3.57) 0.03 Presence of Pl A2 allele 1.55 (1.01 to 2.4) Acute rejection 1.43 (0.94 to 2.2) 0.09 Panel reactive antibodies 1.73 (0.96 to 3.5) % Donor age 1.01 (0.99 to 1.02) 0.28 HLA-DR 1 identity (genomic) 1 (0.7 to 2) 0.30 a CI, confidence interval.

6 2604 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 allograft acute rejection during the first postransplant year. We found a significantly higher prevalence of acute rejection among subjects with at least one Pl A2 allele compared to those without this allele. In addition, the presence of the Pl A2 allele conditioned a higher serum creatinine at discharge and portended a lower 2-yr graft survival. Patients with the Pl A2 allele were at risk of developing acute rejection and suffering a higher number of episodes per patient. This effect was independent of the presence of delayed graft function, a factor that has also been associated with acute rejection in some (16), but not all, studies (17). In addition, the Pl A2 group tended to present biopsy-proven vascular rejection (Banff scores IIb and III) more frequently than patients without the Pl A2 allele (6 of 10 versus 1of8;P 0.065, two-tailed). Although it would be premature to draw any conclusion from these differences at this point, these results are consistent with platelet aggregation being a relevant component of vascular rejections, and underscore the early findings of 111-In-labeled platelet accumulation in vascular rejections (1) and platelet-endothelial aggregates in fine needle aspiration biopsies of severe rejections (2). Many studies have shown that the number of acute rejection episodes is one of the major risk factors for chronic rejection and graft survival (18,19). In the present study, graft survival at 2 yr was lower in those patients showing the Pl A2 allele (Figure 2). In addition, proteinuria 1.5 g/d 1 yr after grafting was more common among patients with the Pl A2 allele (Table 2), suggesting that chronic graft rejection may also be more frequent in these patients. Additional studies including the histologic confirmation of chronic rejection are needed to clarify this point. Patients with the Pl A2 allele showed a significantly higher serum creatinine at discharge than those patients without this allele. This effect was independent of the age of the donor and the presence of delayed graft function. Interestingly, there was an interaction between the presence of delayed graft function and the Pl A2 allele, as these patients showed higher serum creatinine levels at discharge. Serum creatinine at discharge is an accurate indicator of subsequent graft survival and, in the UNOS and UCLA registries, a 7 percentage point drop in 1-yr graft survival was noted with each unit of serum creatinine 2 mg/dl. (20,21). In fact, in the present study, the presence of the Pl A2 allele was associated with a significant 11.5% decrease in graft survival at 2 yr, compared to patients without the allele (Figure 2). In addition to well-known predictors of allograft survival such as serum creatinine at discharge, acute tubular necrosis, and CMV status, glycoprotein IIIa/IIb genotype seems to emerge as a novel genetic marker with prognostic value in renal transplantation, as evidenced by Cox regression analysis of our data. Our findings relating Pl A2 as a potential and independent risk factor for acute rejection might point to new preventive measures. As a matter of fact, it is tempting to hypothesize that patients showing the Pl A2 allele may benefit from the early institution of an antiplatelet drug. The recent finding of a greater inhibition of platelet aggregation by aspirin in these individuals compared to those without the Pl A2 allele favors this hypothesis (22). Aspirin may have the additional protective effect of reduced expression of the endothelial cell adhesion molecules vascular cell adhesion molecule-1 and E-selectin by inhibiting the mobilization of nuclear factor- B (nuclear transcription factor) (23). A similar proportion of Pl A2 and Pl A2 patients received an antiplatelet drug at any time during the course of the study as clinically indicated by the consultant nephrologist (mostly cardiovascular problems). In addition, the percentage of individuals suffering an acute rejection was not different among patients receiving this treatment and those that did not receive the treatment. Finally, when the use of an antiplatelet drug was added in the logistic regression analysis to predict the appearance of an acute rejection episode, the results did not change (data not shown). Thus, it is unlikely that the association of Pl A2 polymorphism and acute rejection reported herein were linked to the use of antiplatelet drugs. However, this information could not be used to infer anything about the potential benefit of a specific trial to address the role of the early institution of this therapy in the prevention of acute rejection. This aspect clearly deserves future investigations. In conclusion, we have demonstrated that the Pl A2 polymorphism is an independent risk factor for acute renal graft rejection affecting short-term graft survival. Future studies aimed at preventing the mechanisms by which this polymorphism induces a hemostatic imbalance favoring platelet aggregation may be important in preventing acute rejection and its impact on chronic rejection. Acknowledgments This study was supported by Grant FIS 96/0857 from the Spanish Ministry of Health (Instituto de Salud Carlos III). References 1. Leithner C, Sinzinger H: Indium-111 labelled platelets in chronic kidney transplant rejection. Lancet ii: , Von Willebrand E, Zola H, Hayry P: Thrombocyte aggregates in renal allografts. Transplantation 39: , Lemstrom K, Koskinen P, Hayry P: Molecular mechanisms of chronic renal allograft rejection. Kidney Int 48: S2 S10, Lages B, Weiss HJ: Evidence for a role of glycoprotein IIb-IIIa, distinct from its ability to support aggregation, in platelet aggregation, in platelet activation by ionophores in the presence of extracellular divalent cations. Blood 83: , Lefkovits J, Plow EF, Topol EJ: Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med 332: , Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffmann T: Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest 83: , Kunicki TJ, Aster RH: Isolation and immunologic characterization of the human platelet alloantigen, Pl A1. Mol Immunol 16: , Newman PJ, Derbes RS, Aster R: The human platelet alloantigens, Pl A1 and Pl A2, are associated with a Leucine 33 /Proline 33 amino acid polymorphism in membrane glycoprotein IIIa, and

7 J Am Soc Nephrol 10: , 1999 Pl A2 Polymorphism in Renal Transplantation 2605 are distinguishable by DNA typing. J Clin Invest 83: , Weiss EJ, Bray PF, Tayback M, Schulman SP, Kickler TS, Becker LC, Weiss JL, Gerstenblith G, Goldschmidt-Clermont PJ: A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. N Engl J Med 334: , Feng D, Lindpaintner K, Larson MG, O Donnell CJ, Lipinska I, Schimitz C, Sutherland P, Muller JE, Levy D, Tofler GH: Increased platelet aggregability associated with platelet GPIIIa Pl A2 polymorphism. Circulation 96: I-412a, Herrero I, Torras J, Carrera M, Castells A, Pasto L, Gil-Vernet S, Alsina J, Grinyo JM: Evaluation of a preservation solution containing fructose-1,6-diphosphate and mannitol using the isolated perfused rat kidney: Comparison with Euro-Collins and University of Wisconsin solutions. Nephrol Dial Transplant 10: , Torres A, Machado M, Concepcion MT, Martin N, Lorenzo V, Hernandez D, Rodriguez AP, Rodriguez A, de Bonis E, Gonzalez-Posada JM, Hernandez A, Salido E: Influence of vitamin D receptor genotype on bone mass changes after renal transplantation. Kidney Int 50: , Solez K, Axelsen RA, Benediktsson H, Burdick JF, Cohen AH, Colvin RB, Croker BP, Droz D, Dunnill MS, Halloran PF, Hayry P, Jennette JC, Keown PA, Marcussen N, Mihatsch MJ, Morozumi K, Myers BD, Nast CC, Olsen S, Racusen LC, Ramos EL, Rosen S, Sachs DH, Salomon DR, Sanfilippo F, Verani R, von Willebrand E, Yamaguchi Y: International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 44: , Gonzalez-Posada JM, Garcia-Castro MC, Tamajon LP, Torres A, Hernandez D, Losada M, Maceira B, Salido E: HLA-DR class II, and ICAM-1 expression on tubular cells taken by fine-needle aspiration biopsy in renal allograft dysfunction. Nephrol Dial Transplant 11: , Zimrin AB, Gidwitz S, Lord S, Schwartz E, Bennett JS, White GC 2nd, Poncz M: The genomic organization of platelet glycoprotein IIIa. J Biol Chem 265: , Canafax DM, Torres A, Fryd DS, Heil JE, Strand MH, Ascher NL, Payne WD, Sutherland DE, Simmons RL, Najarian JS: The effects of delayed function on recipients of cadaver renal allografts: A study of 158 patients randomized to cyclosporine or ALG-azathioprine. Transplantation 41: , Troppmann C, Gillingham KJ, Benedetti E, Almond PS, Gruessner RW, Najarian JS, Matas AJ: Delayed graft function, acute rejection, and outcome after cadaver renal transplantation: A multivariate analysis. Transplantation 59: , Almond PS, Matas A, Gillingham K, Dunn DL, Payne WD, Gores P, Gruessner R, Najarian JS: Risk factors for chronic rejection in renal allograft recipients. Transplantation 55: , Kasiske BL: Clinical correlates to chronic renal allograft rejection. Kidney Int 63: S71 S74, Cecka JM, Terasaki PI: The UNOS Scientific Renal Transplant Registry. Clin Transpl 1992, pp Terasaki PI, Cecka JM, Lim E, Takemoto S, Cho Y, Gjertson D, Ogura K, Koyama H, Mitsuishi Y, Yuge J: UCLA and UNOS Registries: Overview. Clin Transpl 1991, pp Cooke GE, Bray PF, Hamlington JD, Pham DM, Goldschmidt- Clermont PJ: Pl A2 polymorphism and efficacy of aspirin. Lancet 351: 1253, Weber C, Erl W, Pietsch A, Weber PC: Aspirin inhibits nuclear factor-kappa B mobilization and monocyte adhesion in stimulated human endothelial cells. Circulation 91: , 1995