Specificity of protein C and protein S assays

Summary Specific tests for the measurement of protein C antigen and activity and protein S antigen are used in the clinical laboratory for the routine diagnosis of hereditary protein C and protein S deficiency. The performance of these tests is reviewed and discussed. Special attention is paid to the application of these tests for the analysis of patients on oral anticoagulant therapy. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Activated protein C

Summary.  Protein C is a vitamin K-dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to down-regulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor-1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APC's direct effects on various cell types, it is clear that APC's molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells.     

蛋白C和蛋白S活性、活化蛋白C抵抗与急性缺血性脑血管病的相关性研究

目的研究PC和PS活性、APCR与急性缺血性脑血管病间的关系。方法对804例急性脑血管病人及168例健康对照组行PC、PS,APCR检测。结果在缺血性中风组,PC、PS活性分别为115.8%±125%和112.6%±138%明显低于出血性中风组的119.2%±13.3%和112.1%±16.5%,对照组的120.2%±12.8%和122.4%±15.8%,P<0.01。进一步比较发现,<45岁的缺血性中风组的PC、PS分别为54.67%±8.9%,40.49%±9.1%明显低于其他各组,PS在45岁以上的缺血性中风组为119.2%±15.6%±明显低于对照组的122.4%±45 8%,p<0.01,其余各组间无显著差异。APCR总发生率66%(64/972),其中缺血性中风组为7.14%(46/644),出血性中风组为625%(10/160),p>0.05,45岁以下缺血性中风组APCR的发生串46.3%(25/54),明显高于其他各组。结论PC、PS活性降低,APCR是缺血性脑血管病发生的重要因素,特别与45岁以下的缺血性中风的发生密切相关。     

遗传性蛋白C缺陷症家系的一个基因突变

目的 研究一个遗传性蛋白C（PC）缺陷症家系的遗传表型及基因特征。方法 PC活性用凝固法测定，PC抗原用ELISA方法测定。用PCR扩增2代家系12个成员中4个PC活性及抗原减低的PCⅡ-Ⅸ号外显子片段，用单链构象多态性（SSCP）分析cDNA变性后的差异，用测序法检测突变点。用限制性酶切验证突变点，同时分析家系的基因型。结果 该家系2代4名成员PC抗原水平在34．3％－67．8％（参考值80％－120％）。PC活性在22％－49％（参考值70％－130％），较正常参考范围明显减低。限制性酶切分析该家系12名成员时发现9名成员存在基因的突变。基因突变位点在Ⅶ号外显子第6219位核苷酸G→A突变，使正常编码的CGG精氨酸突变为CAG谷氨酰胺。结论 该家系为I型PC缺陷症，基因分析证明先证为杂合子型。在PCⅦ号外显子上第6219位核苷酸G→A突变，在蛋白质合成过程中第169位精氨酸被谷氨酰胺替代（R→Q），为目前国内献中尚未报道的一个基因突变点。     

Impaired secretion of the elongated mutant of protein C (protein C-Nagoya). Molecular and cellular basis for hereditary protein C deficiency.

Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a single guanine residue (8857G) among four consecutive guanine nucleotides [380Trp(TGG)-381Gly(GGT)] in exon IX, which encodes the carboxyl-terminal region of protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino acids (381Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced the third guanine residue upstream from the deletion with cytosine, thereby creating a new AvaI site in an otherwise normal allele. Analysis of the polymerase chain reaction products derived from this mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnormality leads to protein C deficiency, an expression plasmid containing this mutation was transfected into COS 7, BHK, and psi-2 cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA and immunoprecipitation analysis with [35S]methionine labeling indicated that the mutant protein C, which was larger in size than normal, was mostly retained within the cells, and only a small portion of it was secreted into the medium. These results suggest that most of Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift mutation apparently leads to protein C deficiency by impairment of secretion of the elongated protein C into plasma.     

C反应蛋白质粒的构建表达及纯化

目的 在大肠埃希菌中表达和纯化C反应蛋白（CRP），并鉴定纯化蛋白的免疫反应性，为CRP的检测打基础。方法 应用逆转录（RT）PCR方法，从人肝细胞文库中扩增人C反应蛋白cDNA基因片段，与表达质粒载体pCRT7／NTTOPO重组，获得表达质粒CRP—pCRT7／NT。用氨苄青霉素平板筛选转化子，双酶切与DNA测序鉴定CRP基因表达质粒pCRT7／NTTOPO载体的整合状态，用IPTG诱导表达，并经组氨酸亲和层析柱纯化获得CRP的蛋白纯品。采用Western印迹的方法鉴定纯化蛋白的免疫反应性。结果 利用表达载体pCRT7／NTTOPO载体表达的含6个组氨酸标签的CRP，在SDS—PAGE上出现1条相对分子质量约30000的阳性条带，经组氨酸亲和层析柱纯化后用Western印迹杂交证实为目的蛋白。结论 成功地构建了在大肠埃希菌表达CRP的质粒，建立了蛋白纯化的系统，为下一步CRP检测试剂盒的制备打下了基础。     

The protein C pathway and pathologic processes

Summary.  Alterations in expression of protein C (PC) pathway components have been identified in patients with active inflammatory disease states. While the PC pathway plays a pivotal role in regulating coagulation and fibrinolysis, activated PC (aPC) also exhibits cytoprotective properties. For example, PC-deficient mice challenged in septic/endotoxemic models exhibit phenotypes that include hypotension, disseminated intravascular coagulation, elevated inflammatory mediators, neutrophil adhesion to the microvascular endothelium, and loss of protective endothelial and epithelial cell barriers. Further, inflammatory bowel disease has been correlated with diminished endothelial PC receptor and thrombomodulin levels in the intestinal mucosa. Downregulated expression of the cofactor, protein S, as well as PC, is also associated with ischemic stroke. Studies to elucidate further the structural elements that differentiate the various functions of PC will serve to identify novel therapeutic approaches toward regulating these and other diseases.     

Hypercoagulability test strategies in the protein C and protein S pathway

The protein C-protein S pathway is critical in controlling normal clot formation to provide homeostasis between thrombosis and hemorrhage. Abnormalities have been described in which the pathway is altered because factor V cannot be degraded by activated protein C, producing activated protein C resistance. It has been known for nearly two decades that deficiencies in protein C and protein S can result in impaired inhibition of clot formation resulting in increased risk for thrombosis. This article describes the testing strategies associated with the diagnosis of activated protein C resistance, protein C deficiency, and protein S deficiency.     

蛋白C基因新突变His134Asn所致Ⅰ型蛋白C缺乏症

目的：研究一家族性血栓病相关病因的表型及基因型。方法：先证者、其母、二兄分别在３５，１９，３３岁起无明显诱因患反复性下肢深静脉血栓（ＤＶＴ）。对其四代１３个家庭成员的抗凝血酶Ⅲ、蛋白Ｃ（ＰＣ）、蛋白Ｓ（ＰＳ）、纤溶酶原的抗原和活性及活化的ＰＣ抗性等进行检测。结果：该家族５个成员患有Ⅰ型杂合子ＰＣ缺乏症（ＰＣ抗原和活性降低５０％左右）。ＰＣ基因各外显子及外显子、内含子连接区聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）未发现明显的异常带；亚克隆后测序发现ＰＣ的第Ⅵ外显子３４４４Ｃ→Ａ导致１３４Ｈｉｓ→Ａｓｎ变异，这是国外尚未报道的新突变，被命名为ＰＣ长沙。此突变使限制性内切酶ＨｐｈⅠ位点丧失，用ＰＣＲ／ＨｐｈⅠ进行家系分析，证实了６个家系成员（包括５个ＰＣ缺乏者）具有同样的突变。结论：Ｈｉｓ１３４Ａｓｎ是此家族ＰＣ缺乏所致ＤＶＴ的密切相关病理基因型。     

半胱氨酸蛋白酶抑制剂 Ｃ 重组蛋白的制备

摘要：目的 构建半胱氨酸蛋白酶抑制剂 Ｃ（ＣｙｓＣ）原核表达载体，用以快速获取低成本、高浓度、高纯度的 ＣｙｓＣ 纯化蛋白，为 制备应用于临床实验室检测的 ＣｙｓＣ 标准物质和质控品奠定基础。 方法 用 Ｃｏｄｏｎ Ａｄａｐｔａｔｉｏｎ Ｔｏｏｌ 将人的 ＣｙｓＣ 编码序列优化 为大肠杆菌偏好的密码子序列，再将合成的 ＤＮＡ 片段克隆至 ｐＥＴ?２８ａ（＋）原核表达载体。 将重组 ＣｙｓＣ 表达质粒转化至Ｅ． ｃｏｌｉ ＢＬ２１ 感受态中，优化诱导表达条件、确定最优表达条件组合及有效表达序列，并用融合标签对重组蛋白进行纯化。 结果 成 功构建重组 ＣｙｓＣ 原核表达质粒，明确 ＣｙｓＣ 表达序列，确定异丙基硫代半乳糖苷（ＩＰＴＧ）诱导重组蛋白表达浓度、诱导温度等 条件，纯化的 ＣｙｓＣ 蛋白可通过临床检测，且稀释倍数与检测浓度间呈现线性关系。 结论 成功构建重组 ＣｙｓＣ 的高效原核表 达系统，重组 ＣｙｓＣ 蛋白纯度大、浓度高、生产周期短，有望为 ＣｙｓＣ 标准物质和质控品的制备提供物质基础。