Protein C activation in NIDDM patients

Summary Enhanced activation of the clotting system has been recently implicated in the pathogenesis of vascular complications in patients with diabetes mellitus. Abnormalities of the anticoagulant system may constitute a potential trigger factor for the haemostatic activation observed in diabetic subjects. The current study aimed to evaluate anticoagulant activity in diabetic patients by assessing the plasma levels of activated protein C-protein C inhibitor complex; and by measuring the anticoagulant response to exogenous thrombomodulin. This study comprised 61 patients (34 men, 27 women) with non-insulin-dependent diabetes mellitus (NIDDM) of whom 22 showed microalbuminuria and 39 normoalbuminuria. Data obtained in 31 non-obese and non-diabetic subjects were available for comparison. The plasma levels of fibrinogen (p < 0.02), prothrombin fragment 1 + 2 (p < 0.05), fibrin monomer (p < 0.0001), protein C antigen (p < 0.005), total protein S antigen (p < 0.02), soluble thrombomodulin (p < 0.005) and soluble E-selectin (p < 0.005) were significantly higher in diabetic patients than in healthy subjects. The plasma level of activated protein C-protein C inhibitor complex (7.4 ± 3.8 vs 3.0 ± 0.4 pmol/l) was significantly higher (p < 0.0001) and the anticoagulant response to exogenous thrombomodulin (23.4 ± 2.6 vs 35.3 ± 3.0 ng/ml) was markedly lower (p = 0.005) in all diabetic patients than in healthy subjects. Cases with microalbuminuria presented low plasma levels of activated protein C-protein C inhibitor complex (5.5 ± 0.6 vs 8.6 ± 0.7 pmol/l, p < 0.05) and significantly decreased values of the anticoagulant response to exogenous thrombomodulin (16.5 ± 2.9 vs 23.4 ± 2.6 %, p = 0.03) as compared to those with normoalbuminuria. The present study suggests that the hyper-coagulable state in NIDDM is associated with an increased activation of protein C but with a poor plasma reactivity to the anticoagulant effect of thrombomodulin. [Diabetologia (1996) 39: 1455–1461] Received: 27 February 1996 and in revised form: 3 June 1996     

(-)-Adrenaline elicits positive inotropic, lusitropic, and biochemical effects through β2-adrenoceptors in human atrial myocardium from nonfailing and failing hearts, consistent with Gs coupling but not with Gi coupling

Activation of either coexisting β₁- or β₂-adrenoceptors with noradrenaline or adrenaline, respectively, causes maximum increases of contractility of human atrial myocardium. Previous biochemical work with the β₂-selective agonist zinterol is consistent with activation of the cascade β₂-adrenoceptors→Gsα-protein→adenylyl cyclase→cAMP→protein kinase (PKA)→phosphorylation of phospholamban, troponin I, and C-protein→hastened relaxation of human atria from nonfailing hearts. However, in feline and rodent myocardium, catecholamines and zinterol usually do not hasten relaxation through activation of β₂-adrenoceptors, presumably because of coupling of the receptors to Gi protein. It is unknown whether the endogenously occurring β₂-adrenoceptor agonist adrenaline acts through the above cascade in human atrium and whether its mode of action could be changed in heart failure. We assessed the effects of (-)-adrenaline, mediated through β₂-adrenoceptors (in the presence of CGP 20712A 300 nM to block β₁-adrenoceptors), on contractility and relaxation of right atrial trabecula obtained from nonfailing and failing human hearts. Cyclic AMP levels were measured as well as phosphorylation of phospholamban, troponin I, and protein C with Western blots and the back-phosphorylation procedure. For comparison, β₁-adrenoceptor-mediated effects of (-)-noradrenaline were investigated in the presence of ICI 118,551 (50 nM to block β₂-adrenoceptors). The positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline were accompanied by reductions in time to peak force and time to reach 50% relaxation. (-)-Adrenaline caused similar positive inotropic and lusitropic effects in atrial trabeculae from failing hearts. However, the inotropic potency, but not the lusitropic potency, of (-)-noradrenaline was reduced fourfold in atrial trabeculae from heart failure patients. Both (-)-adrenaline and (-)-noradrenaline enhanced cyclic AMP levels and produced phosphorylation of phospholamban, troponin I, and C-protein to a similar extent in atrial trabeculae from nonfailing hearts. The hastening of relaxation caused by (-)-adrenaline together with the PKA-catalyzed phosphorylation of the three proteins involved in relaxation, indicate coupling of β₂-adrenoceptors to Gs protein. The phosphorylation of phospholamban at serine16 and threonine17 evoked by (-)-adrenaline through β₂-adrenoceptors and by (-)-noradrenaline through β₁-adrenoceptors was not different in atria from nonfailing and failing hearts. Activation of β₂-adrenoceptors caused an increase in phosphorylase a activity in atrium from failing hearts further emphasizing the presence of the β₂-adrenoceptor–Gsα-protein pathway in human heart. The positive inotropic and lusitropic potencies of (-)-adrenaline were conserved across Arg16Gly- and Gln27Glu-β₂-adrenoceptor polymorphisms in the right atrium from patients undergoing coronary artery bypass surgery, chronically treated with β₁-selective blockers. The persistent relaxant and biochemical effects of (-)-adrenaline through β₂-adrenoceptors and of (-)-noradrenaline through β₁-adrenoceptors in heart failure are inconsistent with an important role of coupling of β₂-adrenoceptors with Giα-protein in human atrial myocardium.     

Control of cardiac myofilament activation and PKC-betaII signaling through the actin capping protein, CapZ

Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilaments from transgenic mice (TG-CapZ) expressing a reduced amount of CapZ demonstrate altered function and protein kinase C (PKC) signaling [Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, and Solaro RJ., Circ. Res. 90 (2002) 1299-306]. The aims of the current study were to determine the direct effects of CapZ on myofilament function and on PKC signaling to the myofilaments. Our studies compared mechanical properties of single myocytes from TG-CapZ mouse hearts to wild-type myocytes from which CapZ was extracted using PIP(2). We found that myofilaments from CapZ-deficient transgenic myocardium exhibited increased Ca(2+) sensitivity and maximum isometric tension. The extraction of CapZ from wild-type myofilaments replicated the increase in maximum isometric tension, but had no effect on myofilament Ca(2+) sensitivity. Immunoblot analysis revealed that the extraction of CapZ was associated with a reduction in myofilament-associated PKC-beta(II) and that CapZ-deficient transgenic myofilaments also lacked PKC-beta(II). Treatment of wild-type myofilaments with recombinant PKC-beta(II) reduced myofilament Ca(2+) sensitivity, whereas this effect was attenuated in myofilaments from TG-CapZ mice. Our results indicate that cardiac CapZ directly controls maximum isometric tension generation, and establish CapZ as an important component in anchoring PKC-beta(II) at the myofilaments, and for mediating the effects of PKC-beta(II) on myofilament function.     

支气管肺炎患儿血清IGF-Ⅱ、hs-CRP和LT检测的临床应用

目的：探讨了支气管肺炎患儿治疗前后血清IGF-Ⅱ、hs-CRP和LT水平的变化及临床意义。方法：应用放免法、免疫比浊法和酶联法对33例支气管肺炎患儿进行了治疗前后血清IGF-Ⅱ、hs-CRP和白三烯（leukotriene,LT）测定,并与35名正常健康儿做比较。结果：支气管肺炎患儿在治疗前血清IGF-Ⅱ、hs-CRP和LT水平非常显著地高于正常儿组（P〈0.01）,经治疗2周后则与正常儿组比较无显著性差异（P〉0.05）。结论：检测支气管肺炎患儿治疗前后血清IGF-Ⅱ、hs-CRP和LT水平的变化对疾病的诊断、治疗和预后判断均具有一定临床价值。     

人类新基因era基因组织分布的免疫组织化学研究

目的为了研究人era基因这一人类新基因表达产物的组织分布情况.方法根据人era序列设计并合成扩增引物,用PCR法从pUC19-hera质粒中扩增人era基因(h-era)全长cDNA和h-ERAC端的结构域基因,并分别克隆到(His)6融合表达载体pRSET-C和非融合表达载体pDH中,诱导表达(His)6-h-ERA融合蛋白和h-ERAC端结构域蛋白.用h-ERAC端结构域蛋白免疫新西兰白兔,制备兔抗h-ERA抗血清.用Western-b10t对兔抗h-ERA抗血清.进行了鉴定,并成功地制备了兔抗h-ERA血清.利用在大肠杆菌中高表达、纯化的(His)6-h-ERA融合蛋白,对已制备的兔抗人ERA抗血清进行进一步鉴定纯化获得抗h-ERA的特异性多抗;再对4月～5月人胎儿心、肝、肺、脾、肾等脏器和胃、小肠、睾丸、胸腺、胰脏及胆囊进行免疫组织化学染色.结果在人类4月～5个月胎儿心、肝、肺、脾、肾脏等脏器和胃、小肠、睾丸中均有免疫反应阳性产物出现.胰脏、心、肺脏等呈高表达,脾脏、胃、小肠呈中等表达,睾丸、肝脏、肾脏呈低表达,胆囊、胸腺无免疫阳性表达产物出现.结论人ERA在人类正常胎儿不同组织中有表达,但人ERA蛋白在各种组织中分布情况有差异,可能是一种新型功能蛋白.     

支气管肺炎患儿治疗前后血清IGF-Ⅱ、IL-6、IL-8和hs-CRP检测的临床意义

目的：探讨了支气管肺炎患儿治疗前后血清IGF-Ⅱ、IL-6、IL-8和hs-CRP水平的变化及意义。方法：应用放射免疫分析和免疫比浊法对36例支气管肺炎患儿进行了血清IGF-Ⅱ、IL-6、IL-8和hs-CRP测定,并与35名正常健康儿作比较。结果：在治疗前,支气管肺炎患儿血清IGF-Ⅱ、IL-6、IL-8和hs-CRP水平非常显著地高于正常儿组（P〈0.01）,经治疗一周后与正常儿组比较,则无显著性差异（P〉0.05）。结论：检测支气管肺炎患儿血清IGF-Ⅱ、IL-6、IL-8和hs-CRP水平的变化对疾病的诊断、治疗和预后判断均有重要的临床价值。     

辛伐他汀对阿霉素致心衰兔心功能的影响

目的：探讨不同剂量辛伐他汀对阿霉素所致心衰兔保护作用及其抗心衰的机制。方法：雄性新西兰大耳白兔60只,随机数字表法分为正常对照组（CON）、CHF模型组（CHF）、CHF＋辛伐他汀低剂量组（LD-SIM）、CHF＋辛伐他汀中剂量组（MD-SIM）、CHF＋辛伐他汀高剂量组（HD-SIM）。正常对照组（CON）给予耳缘静脉等体积生理盐水,每周1次,共10周。其余组均给予生理盐水注射液稀释的盐酸阿霉素,按4 mg/kg,每周1次,共10次,而辛伐他汀干预组同时给予低、中、高剂量辛伐他汀灌胃治疗,剂量分别为0.3 mg/（kg·d）,1.5 mg/（kg·d）,3.0 mg/（kg·d）。10周后测定大鼠心功能左室肥厚指数、ELISA测定血清中hs-CRP和MMP-13含量。结果：各药物治疗组可不同程度改善左室肥厚,其中辛伐他汀大剂量组与CHF模型组比较差异有统计学意义（P〈0.01）,而辛伐他汀中剂量组与其比较差异有统计学意义（P〈0.05）,而小剂量治疗组与之比较差异无统计学意义（P〉0.05）;大中剂量治疗组心功能指标（＆#177;dp/dtmax）明显优于模型组,差异有统计学意义（P〈0.01）,辛伐他汀大剂量组与中剂量组比较差异无统计学意义（P〉0.05）,而小剂量治疗组与模型组比较差异无统计学意义（P〉0.05）;各药物治疗组能降低血清中CRP、MMP-13含量（P〈0.01或P〈0.05）。结论：辛伐他汀能够预防左心肥厚,降低血浆CRP、MMP-13浓度,从而减缓细胞外基质（ECM）的降解改善心功能,这可能是辛伐他汀抗心衰作用机制之一。     

The relationship between aortic aneurysm sac thrombus volume on coagulation, fibrinolysis and platelet activity

Aim

Abdominal aortic aneurysm (AAA) is associated with chronic mural inflammation and a pro-thrombotic diathesis. It has been suggested that both may be related to biologically active intra-sac thrombus. The aim of this study was to examine the relationship between thrombin generation, fibrinolysis, platelet activity and AAA sac thrombus volume.

Methods

30 patients (29 men) of median (IQR) age 75 (71-82) years with an infra-renal AAA > 5.5 cm in antero-posterior diameter were prospectively studied. AAA, lumen and thrombus volumes were calculated using a CT workstation (Vitrea). Plasma thrombin-antithrombin (TAT), plasminogen activator inhibitor (PAI)-1, and soluble (s) P-selectin were measured as biomarkers of coagulation, fibrinolysis and platelet activity, respectively

Results

Median (IQR) AAA total, lumen and thrombus volumes were 188 (147-247) cm³, 80 (54.3-107) cm³ and 97.6 (63-127) cm³ respectively.TAT levels were significantly higher (median, QR, 7.15 [4.7-31.3] μg/L, p = < 0.001) and sP-selectin levels significantly lower (median, IQR, 80.5 [68-128] ng/ml, p = < 0.0001) than the normal range. PAI-1 levels (median, IQR, 20.9 [8.4-50.7] ng/ml) were normal. There was no correlation between AAA thrombus volume and PAI-1 (r = − 0.25, p = 0.47), sP-Selectin (r = 0.26, p = 0.43) or TAT plasma levels (r = − 0.21, p = 0.54).

Conclusion

The present study confirms that patients with AAA demonstrate haemostatic derangement, but the extent of the haemostatic derangement does not correlate with AAA sac thrombus volume.     

Early identification of acute myocardial infarction by activated protein C--protein C inhibitor complex

INTRODUCTION: Increased coagulation activity due to coronary thrombosis in a ruptured plaque should result in activation of the protein C anticoagulant system with formation of complexes between activated protein C (APC) and the protein C inhibitor (PCI), which reflects coagulation activity. We hypothesized that elevated APC-PCI concentration might allow earlier detection of ongoing myocardial infarction than traditional biochemical markers. We have evaluated a newly devised immunofluorimetric assay for measuring plasma concentration of APC-PCI complexes among patients with suspected acute coronary syndrome. MATERIALS AND METHODS: Blood samples were taken from 340 patients (median 71 years, range 31-97) with suspected acute coronary syndrome at first presentation in the emergency department. Electrocardiogram was recorded and APC-PCI, Troponin I and Creatine kinase-MB concentrations were repeatedly measured 3 times at 6 h interval. RESULTS: The 74 patients who were eventually diagnosed with myocardial infarction had a higher median level of APC-PCI complex than those without myocardial damage; 0.27 vs. 0.20 microg/L (p = 0.001). In a multivariate regression model, APC-PCI level in the fourth quartile (>0.32 microg/L) independently predicted myocardial infarction with an odds ratio of 3.7 (95% CI 1.4-9.6, p < 0.01). CONCLUSION: Early APC-PCI elevation can be detected among patients with a normal first Troponin I and non-ST-elevation myocardial infarction and provides additional risk assessment in acute coronary syndrome.     

Skeletal muscle cells actively shape (auto)immune responses

Histopathological analyses of muscle specimens from myositis patients indicate that skeletal muscle cells play an active role in the interaction with immune cells. Research over the last few decades has shown that skeletal muscle cells exhibit immunobiological properties that perfectly define them as non-professional antigen presenting cells. They are able to present antigens via major histocompatibility complex molecules, exhibit costimulatory molecules and secrete soluble molecules that actively shape the immune response in an either pro- or anti-inflammatory manner. Skeletal muscle cells regulate both innate and adaptive immune responses and are essentially involved in the pathophysiological processes of idiopathic inflammatory myopathies. Understanding the role of skeletal muscle cells might help to identify new therapeutic targets for these devastating diseases. This review summarizes the immunobiological features of skeletal muscle cells, especially in the context of idiopathic inflammatory myopathies, and discusses shortcomings and limitations in skeletal muscle related research providing potential perspectives to overcome them in the future.