Relationship Between Platelet Activation and Acute Rejection After Renal Transplantation

Relationship Between Platelet Activation and Acute Rejection After Renal Transplantation Y. Zhang, X.D. Zhang, L.L. Ma, and D.L. Guan ABSTRACT Objective. To provide an earlier diagnosis and efficiently treat acute rejection episodes (ARE) after renal transplantation, we studied its relationship to platelet activation. Patients and Methods. The peripheral blood levels of platelet surface glycoprotein (CD61), platelet activation-dependent granule membrane protein (CD62p), lysosomal enzyme glycoprotein (CD63), macula densa granule membrane glycoprotein (CD42a), and fibrinogen receptor monoclonal antibody (PAC-1) among 203 patients with uremia in various stages before and after transplantation were assayed by flow cytometry. The patients with ARE were prospectively and randomly assigned to either a treatment group with an antiplatelet activation agent or a control group. Results. The incidence of ARE was remarkably increased among patients with greater expression levels of platelet activation markers in peripheral blood preoperatively. The values of platelet activation markers were significantly higher among patients with ARE compared with those showing either normal graft function or acute tubular necrosis. The greater the increase in CD63, the worse the ARE. The expression levels of platelet activation markers decreased remarkably among the group treated with a platelet activation inhibitor in addition to antirejection therapy: the rejection reversal time shortened and the dose of antihuman thymocyte globulin (ATG) was lower. The sensitivity of platelet activation markers was better than its specificity. Conclusions. Our studies demonstrated an association between platelet activation and ARE after renal transplantation. Platelet activation before transplantation can predict the occurrence of ARE. Platelet activation inhibitor therapy after transplantation improved ARE reversal. HYSIOLOGIC AND PATHOLOGIC evidence have demonstrated that platelet activation plays an important role in the incidence, development, and reversal of an acute rejection episode (ARE) after renal transplantation. Furthermore, platelet aggregation is considered to be an important finding of vascular rejection.1,2 ARE after renal transplantation is associated with several factors that damage vascular endothelium: pharmacologic, mechanical, and hemodynamic. The clotting cascade is activated after platelets adhere to the subendothelial matrix. Fibrin deposition facilitates platelet-platelet aggregation, as well as plateletendothelial and platelet-leukocyte adhesion after transplantation. Endothelial cells, inflammatory cells, and platelets release thromboxanes, platelet activating factors, and plateletderived growth factor, which are crucial mediators of both acute and chronic graft vascular lesion.3 Thus, vascular

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endothelial damage and platelet activation enhance each other causing irreversible graft damage. Direct morphologic evidence also suggests that platelet aggregation to graft endothelial cells is an adverse prognostic sign during the course of rejection.4,5 Recently, a new method has been developed to assay the expression of circulating activated platelet surface glycoproteins using flow cytometry.6,7 Various monoclonal antibodies (MAbs) have been developed to identify activationspecific changes in platelet membranes following activation, From the Department of Urology, Beijing Chaoyang Hospital, Affiliate of Capital Medical University, Beijing, China. Address reprint requests to Yong Zhang, MD, Beijing Chaoyang Hospital, Affiliate of Capital Medical University, No. 8, Baijia Zhuang Road, Beijing 100020, China.

© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.04.006

Transplantation Proceedings, 41, 1547–1551 (2009)

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ZHANG, ZHANG, MA ET AL

including CD61, CD62p, CD63, CD42a, and PAC-1. CD61 is a platelet surface glycoprotein; CD62p is a platelet activation-dependent granule membrane protein; CD63 is a lysosomal enzyme glycoprotein; CD42a is a macula densa granule membrane glycoprotein; and PAC-1 is a fibrinogen receptor monoclonal antibody. In this study, we examined the reactions of corresponding MAbs among blood samples from 203 patients with uremia. We have previously performed studies to demonstrate an association between preoperative platelet activation and postoperative acute renal graft rejection using flow cytometry.8,9 To further investigate this relationship, we increased the number of platelet activation markers from 3 to 6 and patients from 40 to 203, seeking to show a prediction of the occurrence and reversal of ARE, thereby providing theoretical and practical foundations to lower the incidence of ARE, establish an earlier diagnosis, and develop more effective treatments.

nine, a diagnosis of acute tubular necrosis was made based on ultrasound and pathologic tests.

PATIENTS AND METHODS Patient Data

The platelet surface expression levels of CD61, CD62p, CD63, CD42a, and PAC-1 in peripheral blood were assayed in 203 patients with uremia within 4 hours before transplantation. Patients whose renal function returned to normal within 1 week posttransplantation comprised the immediate function group (IGF group). Patients with ARE confirmed by pathologic tests comprised the acute rejection group (ARE group). Patients with acute tubular necrosis confirmed by pathologic tests comprised the acute tubular necrosis group (ATN group). There was no difference among the 3 groups in the following categories: age, gender distribution, duration of dialysis treatment, and graft condition (P ⬎ .05; Table 1). We compared the expression levels of CD61, CD62p, CD63, CD42a, and PAC-1 in patient peripheral blood each postoperative day. Sixty-two patients with ARE based on the biopsy results were randomly and evenly assigned to a treatment or control group. The 2 groups of patients were not different in age, gender distribution, course of disease, duration of dialysis treatment, or graft condition (P ⬎ .05; Table 2). Platelet activation inhibitor, including LipoPGE1 (20 ␮g/d) and low-dose heparin (10 mg/d), were administered to patients in the treatment group where ARE was confirmed. If no bleeding was observed, Lipo-PGE1 and low-dose heparin were administered until rejection reversal. The expression levels of CD61, CD62p, CD63, CD42a, and PAC-1 on peripheral blood platelet surfaces before and after therapy, recovery time of graft function, and 1- and 3-year patient/graft survival rates were compared between the 2 groups.

The study included 203 first kidney transplant patients from January 2002 to December 2004, including 102 men and 101 women of an overall average age of 43.15 ⫾ 21.36 years (range, 24 – 68 years). Among them, 52 patients had diabetic nephropathy; 20, hypertensive nephropathy; 12, polycystic renal disease; and the others had chronic nephritis or nephrotic syndrome. All patients showed normal circulating platelet counts without any blood system complication. All patients had undergone hemodialysis before transplantation: 152, for 9 to 12 h/week; 51, for 6 to 8 h/week. The average length of hemodialysis treatment was 34 months (range, 8 months– 4.5 years). All donors were non– heartbeating with an average warm ischemia time of 6 minutes 41 seconds (range, 4 minutes 48 seconds– 8 minutes 42 seconds), and an average cold ischemia time of 11 hours 35 minutes (range, 3 hours 30 minutes–22 hours). The mismatches of histocompatibility leukocyte antigen (HLA) A, B, and DR types were ⱕ3 with at least 1 DR match. The complement-dependent cytotoxicity test was ⬍10% in all patients. Panel-reactive antibodies were negative (⬍10%) in all patients.

Immunosuppressive Therapy All 203 patients received immunosuppressive therapy with highdose dexamethasone (60 mg) for the first 2 days and 40 mg on day 3 after transplantation in addition to cyclosporine (5– 6 mg/kg/d) or tacrolimus (0.075– 0.1 mg/kg/d), mycophenolate mofetil (1.5 g/d), and prednisone (30 mg/d). The doses of cyclosporine or tacrolimus were increased on day 4 posttransplantation based on the drug concentration. For ARE treatment, methylprednisolone and antihuman thymocyte globulin (ATG) were administered and the dose of cyclosporine or tacrolimus increased.

Diagnosis of ARE and Acute Tubular Necrosis When patients presented with an increased creatinine after operation and a decreased urine volume, a diagnosis of ARE was confirmed by ultrasound and pathologic tests. Sixty-two patients were diagnosed with an ARE: 8 Banff 97 IA; 6 Banff 97 IB; 24 Banff 97 IIA; 14 Banff 97 IIB; and 10 Banff 97 III. When patients presented with decreased urine volume and no increase in creati-

Application of Flow Cytometry to Test Platelet Activation The reagents including mouse antihuman CD61 and CD62p antibody conjugated to fluorescein isothiocyanate (FITC), mouse antihuman CD63 and CD42a antibody conjugated to phycoerythrin (PE), as well as mouse antihuman PAC-1 antibody conjugated to Per-CP were purchased from B.D. Pharmingen (United States). Immunoglobulin from the same species of mouse was used as the control (B.D. Pharmingen) for flow cytometry (Becton Dickinson, FACS Calibur). The levels of CD61 and CD63 were expressed as mean fluorescence intensity. The average fluorescence intensity was used to denote the expression levels of CD61 and CD42a, while the percent positive cells, for CD62p, CD63, and PAC-1. Normative beads produced by American Becton Dickinson Company were used to calibrate the flow cytometry keeping the coefficient of variation (CV) value within 2%.6,7

Study Methods

Table 1. Baseline Characteristics of the 3 Groups Characteristics

IGF Group

ARE Group

ATN Group

No. of patients 101 62 40 Age (y) 42.81 ⫾ 19.74 44.23 ⫾ 20.75 43.99 ⫾ 21.98 Male/female 56:45 34:28 23:17 Duration of dialysis 35.45 ⫾ 22.12 36.90 ⫾ 20.65 33.34 ⫾ 19.91 (months) Warm ischemia 6.22 ⫾ 1.94 6.15 ⫾ 1.79 5.91 ⫾ 1.89 time (min) Cold ischemia time 12.81 ⫾ 9.66 10.96 ⫾ 7.67 11.01 ⫾ 7.94 (hr) HLA mismatches 2.01 ⫾ 0.92 2.12 ⫾ 0.87 2.21 ⫾ 0.71

INHIBITING PLATELET ACTIVATION REVERSED ARE Table 2. Baseline Characteristics of the Treatment Group (n ⴝ 31) and Control Group (n ⴝ 31) Characteristics

IGF Group

ARE Group

Age (y) Male/female Duration of dialysis (months) Warm ischemia time (min) Cold ischemia time (hr) HLA mismatches

43.72 ⫾ 20.33 17:14 36.44 ⫾ 21.09 6.28 ⫾ 1.75 10.93 ⫾ 8.09 2.21 ⫾ 0.79

44.89 ⫾ 20.43 17:14 37.15 ⫾ 20.91 6.01 ⫾ 1.82 10.99 ⫾ 8.67 2.08 ⫾ 0.76

The data for treatment and control groups were compared using Student t test, Kaplan-Meier analysis, correlation and regression analyses, and SPSS for Windows software. This study was approved by our Ethics Committee. All participating subjects provided informed consent.

RESULTS Expression Levels of Platelet Activation in Peripheral Blood Were Related to Kidney Transplant Recovery

Before transplantation, the levels of CD61, CD62p, CD63, CD42a, and PAC-1 in the ARE group (n ⫽ 62) were significantly higher than those in the IGF (n ⫽ 101) or ATN group (n ⫽ 40; Table 3). The 5 levels in the ARE group at the time of diagnosis were significantly higher than those in the IGF group at the time of return of renal function to normal compared with those in the ATN group at the time of that diagnosis (Table 4). Tables 3 and 4 show that the levels of the 5 markers in the IGF and ATN groups were both higher than the preoperative values, although the differences were not significant. Expression Levels of CD63 and PAC-1 Increased Markedly

Tables 3 and 4 show that the expression levels of the 5 markers in the ARE group increased markedly from before the operation to the time of rejection. The expression level of CD61 increased by more than 30%, while those of CD42a and CD62p increased by about 15% to 20%, that of CD63 by 5 to 7 times, and that of PAC-1 by 2 to 4 times. Changing Character of Platelet Activation Markers Upon ARE

The expression level of CD61 increased markedly at 3 days before the diagnosis of ARE. It was the first marker to show an increased level. The amount of PAC-1 increased to a Table 3. Five Markers of the IGF Group, ARE Group, and ATN Group Before Transplantation Markers

IGF Group

ARE Group

ATN Group

CD61 CD63 PAC-1 CD62p CD42a

56.01 ⫾ 10.94 1.74 ⫾ 0.71 2.13 ⫾ 1.29 31.24 ⫾ 5.91 76.12 ⫾ 6.56

78.40 ⫾ 26.14* 15.45 ⫾ 6.55† 9.75 ⫾ 5.49† 41.40 ⫾ 20.42* 91.03 ⫾ 28.73*

65.64 ⫾ 29.37 1.72 ⫾ 1.36 1.99 ⫾ 0.89 33.84 ⫾ 3.96 80.32 ⫾ 19.32

*P ⬍ .05. † P ⬍ .01.

1549 Table 4. Five Markers of the ARE Group (at the Time of Diagnosis), IGF Group (at the Time of Return of Kidney Function to Normal), and ATN Group (at the Time of Diagnosis) Markers

IGF Group

ARE Group

ATN Group

CD61 CD63 PAC-1 CD62p CD42a

62.25 ⫾ 9.33 2.80 ⫾ 0.74 2.84 ⫾ 1.25 35.67 ⫾ 4.76 83.24 ⫾ 3.89

94.25 ⫾ 23.05 17.85 ⫾ 4.35† 11.17 ⫾ 4.67† 50.45 ⫾ 14.86* 99.29 ⫾ 21.84*

†

71.22 ⫾ 21.06 2.85 ⫾ 1.33 3.52 ⫾ 0.79 40.81 ⫾ 3.43 85.69 ⫾ 16.39

*P ⬍ .05. † P ⬍ .01.

greater extent on the day of diagnosis, but this increase was the last to be observed. The slope of the increase in CD63 was steeper than the other 4 markers, while CD63 increased the most (Table 5). Timing Between Increased Levels of Platelet Activation Markers at ARE Occurrence and Kidney Function Recovery

In the ARE group, the level of increase was calculated by subtracting the preoperative expression level from the peak value of CD61, CD62p, CD63, CD42a, and PAC-1. The relativity coefficient (r) was the relative increase in level and the time of ARE reversal. The (r) of CD63 was the greatest, namely .65. Efficiency of ARE Revers by Inhibiting Platelet Activation

Among the patients with ARE, the treatment group (n ⫽ 31) with antiplatelet therapy for 3 days showed markedly lower levels of CD61, CD63, and PAC-1 compared with the control group (n ⫽ 31). Furthermore, the recovery time for graft function was significantly shortened and the dose of ATG decreased compared with the untreated group. Although 1- and 3-year patient and graft survival rates were increased in the treatment group, the differences were not significant upon Kaplan-Meier analysis (P ⬎ .05; Table 6). Specificity and Sensitivity of Platelet Activation Markers

Among patients with ARE after transplantation, 91.9% showed increased CD61, CD62P, CD63, CD42a, and PAC-1 before the operation, and 96.8%, after the operation. In addition, 93.5% displayed a significant decrease in the 5 markers after antiplatelet therapy. The 5 markers of the 20.0% of patients who displayed ATN and 15.8% whose kidney function returned normally, however, experienced comparable increases after the operation. DISCUSSION High Expression Levels of Platelet Activation Markers in Peripheral Blood Before Transplantation Influence Recovery of Renal Transplant Function

The fibrinogen fragments in the blood of patients with uremia can occupy the fibrinogen receptor on the surface of

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ZHANG, ZHANG, MA ET AL Table 5. Changes in CD61, CD62p, CD63, CD42a, and PAC-1 in the ARE Group

Markers

Preoperation

3 Days Before Diagnosis

2 Days Before Diagnosis

1 Day Before Diagnosis

Day of Diagnosis

CD61 CD63 PAC-1 CD62p CD42a

78.40 ⫾ 26.14 13.45 ⫾ 6.55 9.75 ⫾ 5.49 41.40 ⫾ 20.42 91.03 ⫾ 28.73

87.40 ⫾ 20.18 13.51 ⫾ 4.32 9.78 ⫾ 4.24 42.14 ⫾ 17.32 93.40 ⫾ 20.53

91.34 ⫾ 17.82 17.55 ⫾ 3.52 9.97 ⫾ 4.09 46.09 ⫾ 15.62 95.57 ⫾ 14.98

93.27 ⫾ 24.13 17.65 ⫾ 1.38 10.12 ⫾ 1.95 48.74 ⫾ 13.45 97.23 ⫾ 19.42

94.25 ⫾ 23.05 17.85 ⫾ 4.35 11.17 ⫾ 4.67 50.45 ⫾ 14.86 99.29 ⫾ 21.84

platelets preventing them from activation. The platelet aggregation ability in uremia patients is weaker than that in normal people, and their platelets are in the normal functional state. After in vitro dialysis, however, platelet activation and its sensitivity to thrombin increase markedly.10 Therefore, before transplantation, various uremic patients may have a different status of platelet activation that can result in differences in functional graft recovery. Among the 203 cases in our study, the preoperative expression levels of 5 markers on the platelet surface in peripheral blood significantly increased among patients with ARE compared with those showing normal graft function or ATN. However, there were no significant differences among the 5 parameters between patients with normal graft function and those with ATN. This observation suggested that ARE after transplantation might be Table 6. Efficiency of ARE Reversal by Inhibiting Platelet Activation

Day of ARE diagnosis CD61 CD63 PAC-1 CD62p CD42a 1st day after antiplatelet therapy CD61 CD63 PAC-1 CD62p CD42a 3rd day after antiplatelet therapy CD61 CD63 PAC-1 CD62p CD42a Recovery time for graft function (d) Dose of ATG (mg) 1-year patient/graft survival rates (%) 3-year patient/graft survival rates (%) *P ⬍ .05 vs control group. † P ⬍ .01 vs control group.

Control Group (n ⫽ 31)

Treatment Group (n ⫽ 31)

94.34 ⫾ 24.12 16.97 ⫾ 3.87 11.52 ⫾ 4.57 50.25 ⫾ 13.68 98.91 ⫾ 19.64

93.61 ⫾ 25.33 17.55 ⫾ 4.26 12.43 ⫾ 4.36 50.65 ⫾ 12.50 99.89 ⫾ 19.45

92.76 ⫾ 23.44 17.65 ⫾ 4.23 11.66 ⫾ 4.86 52.15 ⫾ 11.54 98.91 ⫾ 19.64

87.16 ⫾ 30.51 15.46 ⫾ 3.40 12.93 ⫾ 5.35 51.56 ⫾ 12.34 96.76 ⫾ 15.81

90.74 ⫾ 26.16 14.38 ⫾ 5.30 12.37 ⫾ 6.79 51.32 ⫾ 14.53 96.61 ⫾ 17.43 18.25 ⫾ 7.30

73.63 ⫾ 14.52* 6.34 ⫾ 2.53† 8.41 ⫾ 5.32* 41.45 ⫾ 15.61* 82.41 ⫾ 18.60* 11.34 ⫾ 4.65*

3600 86.7/80.0

1500† 93.3/86.7

80.0/73.3

86.7/80.0

related to platelet activation before transplantation, whereas ATN after transplantation might not be related to it. This gives us a clue that platelet activation before transplantation has a close relationship to ARE after transplantation. Therefore, by assaying the levels of the 5 markers in patient blood before transplantation to know the state of platelet activation, we might be able to use platelet activation inhibitors suitably before renal transplantation to decrease the incidence of ARE. Changing Character of Expression Levels of Platelet Activation Markers

The expression levels of the 5 markers were significantly higher both before and after transplantation in patients with ARE compared with the IGF and ATN groups. CD61, the GPIIIa part in the GPIIb/IIIa complex,11 is the most abundant platelet plasma membrane glycoprotein; CD63, a lysosomal granule membrane glycoprotein, and PAC-1, a fibrinogen receptor,12 all represent highly activated platelet granule membrane glycoproteins. Lysosomes inside platelets need a stronger stimulant, such as thrombin or collagen in high concentration, to slowly fuse with platelet plasma membranes and release its contents,13,14 thus expressing lysosomal granule membrane glycoproteins on the surface of platelets. A strong stimulant can also release the GPIIb/ IIIa complex changing the spatial structure of the GPIIb/ IIIa complex to reveal the fibrinogen binding site.15 Therefore, the fact that the expression levels of lysosomal granule membrane glycoproteins and fibrinogen receptor monoclonal antibody increased remarkably again demonstrated that platelets were highly activated, which had been shown by morphologic changes observed by electron microscopy. Platelet activation can enhance adherence to endodermal cells, producing aggregation and adherence to leukocytes after transplantation. Then endodermal cells and lymphocytes are activated releasing factors that produce a kidney vascular thrombus, causing ARE. High expression levels of platelet activation markers in peripheral blood obey some rules during ARE: CD61 increases first, CD63 increases most, and PAC-1 increases last. CD61, rising first, is helpful for an early diagnosis of platelet activation and ARE. PAC-1, rising last, is helpful to evaluate the time of platelet activation and to estimate the severity of injury caused by platelet activation. CD63, rising most, not only is easy to monitor, but also assists in the prediction of ARE outcome, for the largest value of the correlation coefficient is the reversal time.

INHIBITING PLATELET ACTIVATION REVERSED ARE

Inhibiting Platelet Activation Has Remarkable Efficacy in ARE Reversal

Among the 62 patients with ARE, the immune inhibitor therapy was the same; the difference was the antiplatelet therapy after definitive diagnosis of ARE: Lipo-PGE1 and low-dose heparin. The results were: on the third day of anticoagulant therapy, the 5 markers decreased remarkably and graft function recovered faster among the treatment group. Thus, without an increased bleeding risk or injury to the liver or kidney, adding antiplatelet drugs in addition to strong immune inhibitor may be of great significance for the prognosis of an ARE. There are many antiplatelet drugs in clinical application, such as aspirin, heparin, and the more recently developed platelet GPIIb/IIIa receptor antagonists (7E3 humanderived monoclonal antibody).16 However, since patients undergoing renal transplantation take prednisone, additional aspirin may increase the risk for alimentary canal bleeding. Patients after surgery also cannot take high doses of heparin. Therefore, Lipo-PGE1 will play an important pharmacologic role to inhibit platelets. Lipo-PGE1 is one type of Lipo-prostaglandin injection, which inhibits platelet aggregation, prevents the formation of microthrombi, protects cell membranes, stabilizes lysosomal membranes, enhances erythrocyte deformation, and improves microcirculation.17 The degradation of fibrin can inhibit platelet aggregation and prevent the formation of microthrombi to improve microcirculation, and lessen or prevent the rise of blood stickiness. They may promote recovery of graft function by preventing platelet activation during ARE. Specificity and Sensitivity of Platelet Activation Markers

From the data of patients in this group, we discovered that these 5 platelet activation detection markers showed high sensitivity to predict the incidence of ARE after renal transplantation, especially when ARE occurred. Almost all of these 5 markers increased remarkably, which was of great significance for the clinical diagnosis of ARE. However, their specificity was not as good as their sensitivity. Considering the great damage ARE may cause, the sensitivity is, of course, more important than specificity of diagnostic markers. Value Ranges of Markers in the Platelet Activation Detection System

According to the data in this group, we recommend marker ranges as such: without platelet activation, the expression level range of CD61 was about 63 ⫾ 10; 35 ⫾ 5 for CD62p; 2.3 ⫾ 0.5 for CD63; 2.8 ⫾ 1.0 for CD42a; and 2.8 ⫾ 1.0 for PAC-1. With platelet activation at the occurrence of ARE,

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the expression level range of CD61 increased by about 30% to 35%, CD42a and CD62p increased by about 15% to 20%, CD63 increased by 5- to 7-fold, and PAC-1 increased by 2- to 4-fold. In conclusion, platelet activation markers can predict the incidence and reversal of ARE after renal transplantation. We evaluated the efficacy of inhibiting platelet activation for reversal of acute rejection. REFERENCES 1. Bustos M, Saadi S, Platt JL: Platelet-mediated activation of endothelial cells: implications for the pathogenesis of transplant rejection. Transplantation 72:509, 2001 2. Meehan SM, Limsrichamrem S, Manaligod JR, et al: Platelets and capillary injury in acute humoral rejection of renal allografts. Hum Pathol 34:533, 2003 3. Abrams CS: Intracellular signaling in platelets. Curr Opin Hematol 12:401, 2005 4. Breddin HK: Can platelet aggregometry be standardized? Platelets 16:151, 2005 5. von Hundelshausen P, Petersen F, Brandt E: Platelet-derived chemokines in vascular biology. Thromb Haemost 97:704, 2007 6. Amabile N, Guerin AP, Leroyer A, et al: Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol 16:3381, 2005 7. Angiolillo DJ, Femandez A, Bemdrao E, et al: Platelet function profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes 54:2430, 2005 8. Zhang Y, Guan DL, Xia CQ, et al: The relationship between the peripheral blood of CD61, CD63, PAC-1 and the transplant kidney function. Transplant Proc 35:1360, 2003 9. Zhang Y, Guan DL, Xia CQ, et al: Clinical study on Lipo PGE1 inhibiting platelet activation in AR after kidney transplantation. Transplant Proc 37:2408, 2005 10. Fogari R, Zoppi A: Is the effect of antihypertensive drugs on platelet aggregability and fibrinolysis clinically relevant? Am J Cardiovasc Drug 5:211, 2005 11. Chiang TM: Recent development of peptides from glycoproteins IIb (alphaIIb) and IIIa (beta3) that inhibit platelet fibrinogen binding. Curr Med Chem Cardiovasc Hematol Agents 3:99, 2005 12. Golino P, Loffredo F, Riegler L, et al: Novel antithrombotic strategies in cardiovascular diseases. Curr Opin Invest Drugs 6:298, 2005 13. Rumbaut RE, Slaff DW: Microvascular thrombosis models in venules and arterioles in vivo. Microcirculation 12:259, 2005 14. Chen J, Lopez JA: Interactions of platelets with subendothelium and endothelium. Microcirculation 12:235, 2005 15. Rother RP, Bell L, Hillmen P, et al: The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. JAMA 293:1653, 2005 16. Kondo K, Umemura K: Clinical pharmacokinetics of tirofiban, a nonpeptide glycoprotein IIb/IIIa receptor antagonist: comparison with the monoclonal antibody abciximab. Clin Pharmacokinet 41:187, 2002 17. Colwell JA: Antiplatelet agents for the prevention of cardiovascular disease in diabetes mellitus. Am J Cardiovasc Drugs 4:87, 2004