血漿凝血酶調(diào)節(jié)蛋白檢測的臨床研究

1、血漿凝血酶調(diào)節(jié)蛋白檢測的臨床研究    【摘要】 為了探討血漿凝血酶調(diào)節(jié)蛋白（PTM）檢測的臨床價值，用ELISA法測定979例患者的PTM，并選擇60名健康人作為對照。結(jié)果表明：對照組PTM 水平為20.40±7.72 g/L，無性別和年齡差異。在疾病組中，原發(fā)性慢性腎小球疾病腎功能衰竭（CRF）組PTM水平高于無CRF組，敗血癥組PTM水平高于非敗血癥組，多臟器功能衰竭（MOF）組PTM水平高于無MOF組（P<0.01）；以>70、>50和>40 g/L為標準，分別預示CRF、敗血癥和MOF的靈敏度為85.7%、86

2、.6%和77.8%，特異性為82.4%、89.5%和77.3%，陽性預示值為77.8%、76.5%和73.7%。系統(tǒng)性紅斑狼瘡（SLE）尿蛋白陽性組PTM水平高于陰性組；糖尿病并發(fā)癥組的PTM水平高于無并發(fā)癥組，并發(fā)微血管病變組的PTM水平高于大血管病變組（P均<0.01）；以PTM高于正常上限值（>35.54 g/L）為標準，預示SLE尿蛋白陽性臨床腎損害、糖尿病并發(fā)癥和微血管病變的靈敏度為77.8%、53.4%和71.2%，特異性為92.3%、97.1%和97.1%，陽性預示值為93.3%、98.6%和97.9%。急性白血?。ˋL）和多發(fā)性骨髓瘤（MM）初診 時PTM升高，兩病

3、并發(fā)腎衰時極度升高（P<0.01）。動態(tài)檢測多發(fā)傷、腦卒中急性期和恢復期、AL和MM化療前后、癌癥術(shù)前后PTM水平與病情變化相關(guān)。以微血管病變?yōu)橹饕膊〉腜TM 水平高于大血管病變疾病 （P<0.01）， 以高于正常上限值為標準，微血管病變疾病的靈敏度為77.7%、特異性71.2%，陽性預示值75.6%。結(jié)論：PTM水平是評估微血管病變疾病的良好指標，也是預警或評估疾病嚴重程度及其演變或療效觀察的有用指標。    【關(guān)鍵詞】 血漿凝血酶調(diào)節(jié)蛋白 疾病嚴重程度/并發(fā)癥 微血管病變 大血管病變    Thr

4、ombomodulin (TM), a thrombin?binding glycoprotein expressed on the endothelial cell surfaces in various tissues, is involved in negative regulation of coagulation through the activation of protein C1. The soluble form of TM, which was arised by proteolytic cleavage from membrane TM on endothelial ce

5、lls, can be detected in human plasma and urine. Since the plasma TM concentration is elevated in a variety of diseases accompanied by endothelial injury, soluble TM is believed to be a good indicator of endothelial damage or activation2,3. Plasma TM is not specific for diagnosis of single disease, b

6、ut it may be helpful to demonstrate the presence of microangiopathy and to assess the severity or complication of diseases.    Materials and Methods    Patients and healthy controls    979 patients admitted to the second hospital of Zhejian

7、g University from 1996 to 2004 were included in this study. The patients were 542 men and 437 women with mean age of 47.6±18.5 years old (range, 15 to 91 years). Sixty healthy volunteers with mean age of 38.6±16.2 years old (range, 12 to 77 years) were included in control group. The sex an

8、d age of the healthy volunteers, the characteristics of the patients including disease stage, complications and severity are summarized in table 1 and 2.    Methods    The levels of plasma TM were measured by enzyme?linked immunosorbent assay (ELISA) kit accor

9、ding to manufacture s instruction (STAGO, France). Venous blood was collected into 3.2% sodium citrate (9 parts of blood 1 part of anticoagulant) using a 10?ml syringe with a 21?gauge needle. Plasma was obtained by centrifuging the blood at 1 500× g for 30 minutes. The plasma samples were froze

10、n and stored at -70 until the assay.    Statistical analysis    The results were expressed as mean±SD, and POMS statistical package was used for multivariate analysis, students t test and correlation analysis. Statistical significance was defined as P val

11、ue<0.05 for a two?tailed test.    Results    PTM level in control group    As shown in Table 1, the PTM level in healthy control group was 20.40±7.72 g/L, no significant difference was observed between different age and sex. Here we

12、 defined PTM level higher than its normal upper limit (1.96 ±SD, i.e. 35.54 g/L) as PTM positive criterion, and the positive rate of the control group was less than 5%.    Table 1. PTM levels of the different sex and age in healthy controls（±SD）    n

13、PTM（g/L） female2521.16±8.33male3519.86±7.3325 years old1819.50±8.2926-45 years old2020.35±7.7846-77 years old2221.41±7.60    PTM levels in relation with the severity and complications of diseases    Cardiocerebral vascular disease The

14、diagnosis was made based on the criteria proposed at the 4th National Conference on Cerebrovascular Disease. Cerebral infarction within 3 days showed significant higher PTM level than the transient ischemic attack (P<0.01), but only 32.1% of the PTM were positive, similar to those of cerebral hem

15、orrhage (33.9%) and coronary heart disease (12.9%). In patients with essential hypertension, PTM level is correlated with hypertension stages (Table 2). Among 137 patients suffering from stroke, 27 were accompanied by diabetes, 28 by hypertension or coronary heart disease, 6 by renal diseases and 76

16、 without underlying diseases, PTM levels of these patients were 31.37±12.06 g/L, 32.47±12.08 g/L, 60.00±23.52 g/L and 31.51±14.13 g/L respectively. Patients with renal diseases had significantly higher PTM than other patients(P<0.01). No correlation was found between PTM and b

17、lood glucose (r=0.0360), cholesterol (r=-0.0587) and triglyceride (r=0.0542) among 280 cardiocerebral vascular diseases or diabetes. PTM was not correlated with fibrinogen (r=0.087) and fibrin degradation product (FDP) (r=0.026) in 81 cases (P>0.05). 26 patients with cerebral infarction and 22 pa

18、tients with cerebral hemorrhage for at least 14±1 days had been followed up PTM dropped when the disease was remitted (cerebral infarction: 31.67±11.50 g/L vs 23.08±9.37 g/L, P<0.01, and cerebral hemorrhage: 33.61±7.24 g/L vs 27.94±5.83 g/L, P =0.02).   

19、60;Primary chronic glomerular disease The diagnosis of primary chronic glomerular disease was confirmed by clinical and/or pathological findings. Patients with chronic renal failure (CRF) had significantly higher PTM level than those without CRF (Table 2). 100% and 55.9% of their PTM were positive r

20、espectively. PTM over 70 g/L would be considered as a CRF risk factor with sensitivity 85.7%, specificity 82% and positive predictive value 77.8%. PTM was found to positively correlated with blood urea nitrogen (BUN) (r=0.5926) and serum creatinine (Scr) (r=0.4781) in 105 patients, blood (2?microglo

21、bulin (2?MG) (r=0.5559) and urine (2?MG (r=0.4409) in 62 patients. There was negative correlation between PTM and creatine clearance rate (Ccr) in 64 patients (r=-0.5844, P<0.01).    Infections PTM level of patients with septicemia was higher than that of patients without sept

22、icemia (Table 2), PTM>50 g/L suggested presence of septicemia with sensitivity 86.6%, specificity 89.5%, and positive predictive value 76.5%. There was no correlation between PTM level and leukocyte or neutrophil count (r=0.2543 and 0.2333, P>0.05).    Multiple trauma PTM l

23、evel of patients with multiple trauma was higher than that of healthy control. These patients with multiple organ failure (MOF) had significantly higher PTM level than those without MOF (Table 2). It was found in 36 patients followed?up that high PTM level dropped when remission was achieved in 20 p

24、atients (23.63±7.20 g/L vs 34.64±10.10 g/L, P<0.01),but it rose higher when disease got worse in 11 patients (54.83±21.20 g/L vs 40.64±15.90 g/L,P<0.01).It was extremely high in 5 patients with disseminated intravascular coagulation (DIC) (126.25±62.06 g/L).  

25、;  Systemic lupus erythematosus (SLE) The diagnosis was made according to the criteria (1982 edition) of American Rheumatism Association among 60 patients with primary or relapsed SLE. The patients with albuminuria had obviously been higher PTM than those without albuminuria (Table 2). PTM

26、 positive indicated renal insufficiency with sensitivity 77.8%,    Table 2. PTM levels of the severity, complication and stage in different diseases（±SD）    Group    nPTM（g/L） Healthy control6020.40±7.72 Macroangiopathy coronary h

27、eart disease3128.44±11.49* cerebral infarction7829.44±14.17*    cerebral hemorrhage5934.02±12.19* essential hypertension7432.91±18.99*    stage2943.00±25.69*    stage2429.41±8.33*    stage21

28、22.95±6.78 transient ischemic attacks1323.23±7.67Microangiopathy Primary chronic glomerular disease11781.54±56.54*    with renal failure49124.10±61.47*    without renal failure6851.13±35.68* Multiple trauma4041.30±16.30* 

29、0;  multiple organ failure1848.70±18.71*#    without multiple organ failure2236.33±11.32* Infection    septicemia1579.93±29.17*#    nonsepticemia3835.52±14.61* Systemic lupus erythematosus62 41.77±34.04*&#

30、160;   with albuminuria3655.81±38.49*×    without albuminuria2622.26±8.55Other diseases Diabetes mellitus14633.25±12.43*    without complication3522.11±6.58    with complication11138.75±11.72*

31、15;×    microangiopathy66 43.10±13.28*§    macroangiopathy4531.89±8.25*    Cancer18833.47±14.25*    metastasis86 41.68±16.96*§§    nonmetastasis10226.54±11

32、.14* Hematologic malignancies    acute leukemia(AL)6535.78±17.21*    multiple myeloma(MM)3536.14±10.05*    with renal failure18105.33±51.35*    *P<0.01，*P<0.05 vs healthy controls；P<0.01 vs stage

33、II and I；P<0.01 vs without renal failure；# P<0.01 vs without multiple organ failure； # P<0.01 vs nonsepticemia；×P<0.01 vs without albuminuria；××P<0.01 vs without complication；§P<0.01 vs macroangiopathy；§§P<0.01 vs nonmetastasis；P<0.01 vs with ren

34、al failure    specificity 92.3% and positive predictive value 93.3%. BUN, Scr and 2?MG were positively correlated with PTM levels (r=0.4913, 0.6368, 0.8641, 0.8251, P<0.01) .    Diabetes The diagnosis was made based on 1997    PTM levels

35、 in microangiopathy and macroangiopathy    The macroangiopathy including cardiovascular and cerebrovascular diseases showed mild increase of PTM, the average PTM level of macroangiopathy was less than 35 g/L. However, in diseases characterized by microangiopathy such as chronic g

36、lomerular disease, systemic lupus erythematosus (SLE) with albuminuria, septicemia and multiple organ failure (MOF), disseminated intravascular coagulation (DIC) and diabetic microangiopathy, PTM level moderately increased with an average level higher than 40 g/L. There was significant difference be

37、tween micro? and macroangiopathy (Table 2). If PTM level was defined higher than the normal upper limit (35.54 g/L) in microangiopathy, the sensitivity was 77.9%, the specificity of 71.4% and the positive predictive value of 75.6%.    Discussion    Previous st

38、udies showed that there was PTM level variation among population of different ages and sex2, but the difference was not demorstrated in our study. PTM is regarded as a reliable marker for endothelial injury, but little is known about its clinical significance. We studied various diseases and found t

39、hat the evelation of PTM was well correlated with the disease severity or the incidence of complications. In terms of predicting sensitivity, specificity and positive predictive value, PTM in CRF, SLE with renal damage and septicemia was high, but PTM in DM with complication showed less sensitivity.

40、 Uehara et al4 consider that a fluctuation in the blood glucose level is significantly related to vascular endothelial cells damage in DM. Therefore, PTM can be considered as a promising marker to evaluate disease progression or complications. However, different PTM limits were used for different di

41、seases; usually PTM exceeding its normal upper limit (35.54 g/L) would indicate endothelial damage. In addition, PTM over 40 g/L may imply disease progression or complications, and PTM over 70 g/L indicates the presence of critical conditions such as CRF, DIC and septicemia. The PTM levels were foll

42、wed?up in some diseases and found that high PTM would drop at remission; or it would rise again when relapsed. It was regarded that the PTM might be a useful index for reflecting the severity and evaluating the curative effect in different diseases. Some reported that TM was also involved in develop

43、ment of DM nephropathy5; PTM could be better than the other makers in indicating diabetes albuminuria6; PTM level reflected activity of rheumatiod arthritis and could be helpful in monitoring disease status and response to therapy7. PTM obviously dropped and the survival periods were prolonged after

44、 therapy in mouses with sepsis8. High level of PTM indicated high death rate in patients with severe acute respiratory syndrome (SARS)9. Dynamic monitoring of plasma TM would be useful in reflecting the severity and evaluating therapeutic response of thrombotic thrombocytopenic purpura (TTP). &

45、#160;  PTM is well correlated with renal function, both primary and secondary CRF or nephropathy show significant PTM increase, elevated PTM may be a response to both glomerular capillary injury and azotemia. Patients with cerebrovascular disease are often accompanied by diabetes, hyperten

46、sion, coronary heart disease. Our study demonstrated that PTM of cerebrovascular disease was not affected by occurrence of DM, hypertension or coronary heart diseases, and was different from these three disease. Neutrophils and their products may interact with endothelium and lead to PTM release10,

47、no association was found between the white blood cell or neutrophils and PTM in patients with infections. While in patients with AL and MM, positive correlation was demonstrated between PTM with blast or plasma cells in bone marrow, indicating that infiltration by blast cells may cause endothelium d

48、amage and result in PTM increase. PTM level was found nothing to do with blood fattiness and glucose in patients wi 2Blann AD, Daly RJ, Amiral J. The influence of age, gender and ABO blood group on soluble endothelial cell markers and adhesion molecules. Br J Haematol，1996; 92: 498-5003Yokota H, Naoe Y, Nakabayashi M, et al. Cerebral endothelial injury in severe head injury: the significance of measurements of serum thrombomodulin and t