Studies on the profibrinolytic actions of heparin and its fractions

Utilizing modified immunochemical methods (ELISA and radioimmunoassay) tissue plasminogen activator, B beta 15-42 RPs and protein C antigen levels were measured in man and a subhuman primate model after subcutaneous and intravenous administration of various low molecular weight heparin fractions. A wide scatter in the data was observed in the t-PA and B beta 15-42 RP levels; however, statistical analysis of the data revealed that certain low molecular weight heparin fractions increased the levels of these endogenous markers of fibrinolysis. No significant alteration in the protein C levels was noted at any time; however, a wide scatter in these data was also evident. The profibrinolytic actions of low molecular weight heparin fractions may be related to the release of t-PA, which is easily measured in plasma. Since it has strong affinity for endogenous sites (thrombus, surface), wide scatter in the data may occur. Physical exercise may also increase the levels. Most of the results reported in our studies represent data on blood samples that were obtained using the simple venipuncture method. We also find that the intravenous administration of the low molecular weight heparin fractions also caused a shortening of the ELT. Since the low molecular weight heparin fractions are heterogeneous in nature, the profibrinolytic actions may be related to one or more of these constituent fragments. Thus, the molecular identity of the profibrinolytic component of low molecular weight fractions remains unknown at this time. Also unknown is if there is a relationship between this effect and anticoagulant activity.     

Expression and localization of the serine proteases high-temperature requirement factor A1, serine protease 23, and serine protease 35 in the mouse ovary

Proteolytic degradation of extracellular matrix components has been suggested to play an essential role in the occurrence of ovulation. Recent studies in our laboratory have indicated that the plasminogen activator and matrix metalloproteinase systems, which were previously believed to be crucial for ovulation, are not required in this process. In this study we have used a microarray approach to identify new proteases that are involved in ovulation. We found three serine proteases that were relatively highly expressed during ovulation: high-temperature requirement factor A1 (HtrA1), which was not regulated much during ovulation; serine protease 23 (PRSS23), which was down-regulated by gonadotropins; and serine protease 35 (PRSS35), which was up-regulated by gonadotropins. We have further investigated the expression patterns of these proteases during gonadotropin-induced ovulation in immature mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly expressed in granulosa cells throughout follicular development and ovulation, as well as in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles, and it was expressed in the ovarian stroma and theca tissues just before ovulation. PRSS35 was expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data suggest that HtrA1 and PRSS35 may be involved in ovulation and CL formation and regression, and that PRSS23 may play a role in follicular atresia.     

Lack of correlation between heparin dose and standard clinical monitoring tests in treatment with unfractionated heparin in critically ill children

The activated partial thromboplastin time (aPTT) and anti-Xa activity are used for monitoring unfractionated heparin (UFH) therapy in children and may not be optimal. OBJECTIVE: Determine correlations of aPTT, anti-Xa and UFH dose in children. Single centre prospective cohort study in children receiving UFH. The aPTT and anti-Xa results from routine coagulation monitoring were collected. Thirty-nine children (median age 18 days) were enrolled. There was no relationship between aPTT and UFH dose (r2=0.12) or anti-Xa and UFH dose (r2=0.03) or aPTT and anti-Xa (r2=0.22). aPTT and anti-Xa do not accurately monitor UFH therapy in children.     

An objective assessment of the interaction of heparin and its fractions with human platelets

We have shown that heparin and heparin fractions cause in vitro platelet aggregation in a large portion of a normal population. Furthermore, this aggregation occurs in a concentration-dependent manner and is not related to the anti-Xa activity of heparin or its fractions. In addition, it appears that at least part of the mechanism by which heparin induces aggregation is through the production of thromboxane. However, this is not the sole mechanism, since approximately 20% aggregation still occurs when thromboxane production is totally inhibited or the thromboxane receptor is completely blocked. Furthermore, although protamine (at the concentrations used) completely neutralizes the anticoagulant activity of heparin, it does not always completely inhibit the platelet aggregating activity of heparin. Finally, we have shown that heparin alone promotes thromboxane production and PF4 release in a whole blood system. Additional studies are needed to characterize further the mechanisms of heparin-induced platelet aggregation.     

粪肠球菌丝氨酸蛋白酶基因突变株的构建和致病性研究

目的 构建粪肠球菌丝氨酸蛋白酶基因（sprE）突变株，并研究sprE基因的功能。方法 用自杀质粒pTX4577构建粪肠球菌基因重组自杀质粒pCQ001，通过体内同源重组，筛选获得sprE基因的突变株，体外研究不同温度和氧化条件对突变株生长能力的影响，通过小鼠腹膜炎和兔心内膜炎模型来研究突变株的毒力下降情况。结果 经同源重组，利用卡那霉素抗性筛选，PCR、脉冲场电泳和Southern印迹进行鉴定获得sprE基因突变株，命名为＊sprE，突变株在40℃的生长能力及在氧化条件下的存活率均明显低于野生株。在小鼠腹膜炎模型中，存活率明显高于野生株，差异有统计学意义（P〈0．01），引起的兔心内膜炎也较野生株轻，差异有统计学意义（P〈0．01）。结论 sprE基因突变株＊sprEgg建成功，sprE基因在粪肠球菌致病中起重要作用，可能是粪肠球菌的毒力因子之一。     

A modified stasis thrombosis model to study the antithrombotic actions of heparin and its fractions

The original stasis thrombosis model of Wessler has been modified. Numerous thrombogenic agents were evaluated for their pathophysiologic effects and were classified in terms of stasis clot in the jugular vein. Alterations produced in coagulation parameters, such as the PT, APTT, thrombin time, activated recalcification time (Hemachron), and thrombelastographic pattern were recorded. Since the pathophysiologic activation of the hemostatic system varies considerably in different diseases, a proper animal model along with a proper type of thrombogenic trigger should be carefully selected to produce pathogenesis and to study the therapeutic responses of heparin and its derivatives. In the modified stasis thrombosis model, besides monitoring the formation of the jugular vein stasis clot, it is proposed that the following tests may be useful to establish hypercoagulable states: Functional levels of various coagulation factors, platelet counts, fibrinogen levels, and whole blood activated clotting times. The nature of activation processes in each thrombogenic challenge should be carefully analyzed in terms of pathways involved; for example, the administration of heterologous serum (such as human, monkey) to rabbits produces anaphylactoid reactions, including hemolysis, thrombocytopenia, clinical chemistry abnormalities (enzymes), and many problems that may involve the complement and immune systems. All previous data obtained using heterologous sera as a thrombogenic trigger are of questionable value as to the efficacy of some of the antithrombotic agents tested against it. In addition to the species and the thrombogenic challenge, the following factors may contribute significantly to the pathophysiologic response and its alteration by various agents: (1) Composition of the thrombogenic agent; (2) effect of preparatory drugs, such as anesthetics, on the hemostatic parameters; (3) alterations on injection time, volume, osmolarity, and temperature; (4) variations in the circulation time of the thrombogenic agent and stasis time of the ligated jugular vein stasis segment; and (5) blood sample collection and handling. Since the kallikrein-kinin cascade is closely associated with the coagulation and the fibrinolytic network, a systemic monitoring of blood pressure may provide information on the effect of thrombogenic agents on hemodynamics.(ABSTRACT TRUNCATED AT 400 WORDS)     

肝素短期应用的抗肝纤维化疗效评价

为探讨肝素对乙型肝炎肝纤维化的影响 ,将 75例慢性乙型肝炎患者随机分为 A、B、C组 ,分别给予常规治疗、肝素 /低分子量肝素和 α-干扰素治疗。治疗前后分别检测血丙氨酸氨基转移酶 (AL T)、凝血酶原时间(PT)、总胆红素 (TBIL)、透明质酸 (HA)和 型胶原 ( - C)水平。 A组和 C组分别有 3例于治疗前后行肝活检术 ,B组中 7例分别于治疗前后行肝活检术。结果治疗后 A、B组 AL T、TBIL水平较治疗前显著下降 (P<0 .0 5 ) ,C组 AL T较治疗前略有升高 ;B组治疗后 HA、 - C水平显著下降 (P<0 .0 5 ) ,而 A、C两组 HA、 - C反较治疗前有所升高 (P>0 .0 5 ) ;A、C两组治疗前后肝纤维化程度无明显变化 ,B组治疗前肝组织胶原纤维增生显著 ,治疗后明显减轻。结论 :肝素可抑制乙型肝炎肝组织中胶原纤维增生 ,α-干扰素短期应用无明显的抗肝纤维化作用     

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.     

Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity.

Antithrombin-III-Stockholm is a new structural variant of antithrombin-III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT-III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha-thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.     

A primate model (Macaca mulatta) to study the pharmacokinetics of heparin and its fractions

We have extensively studied the hemostatic parameters and the responses to the anticoagulant action of heparin and its fractions in the primate model (M. mulatta) and found these to be identical to those obtained in humans. The functional properties of antithrombin III, alpha 2-antiplasmin, and platelet factor 4 were also identical to humans in amidolytic and coagulant assays. Human antibodies against FPA, B beta 15-42 peptide, platelet factor 4, and thromboxane B2 reacted with the primate antigen, and assays were developed to measure these parameters in primates. Infusion of activated prothrombin complex concentrates (more than 100 U/kg/day) on a continual basis up to 3 days resulted in a hypercoagulable state manifested by an elevation of FPA, thromboxane B2, and changes in the thrombelastographic patterns. Similarly, infusion of homologous primate serum also resulted in a hypercoagulable state, as was evident by a sharp increase in the FPA levels. The antithrombotic effects of intravenous and subcutaneous administration of heparin, its low molecular fraction, and their constituents were studied after intravenous and subcutaneous injections. The low molecular weight fractions showed the most effective antithrombotic effects, whereas somewhat milder protection was observed with the starting material and highly anionic fraction. The prolongation of global tests, such as the APTT, TT, and changes in the thromboelastogram did not correlate with the reduction in the blood markers of hypercoagulable state. A modified simplate bleeding time method was used to study the effect of heparin and its fractions on the bleeding profile of heparin fractions. The components of fibrinolytic systems were also measurable in both the clot-based and amidolytic methods to predict the profibrinolytic actions of heparin fractions in its mode. These studies suggest that plasma markers, such as the platelet release proteins, products of thrombin activation, and prostaglandin metabolites, may provide better indices in the monitoring of the antithrombotic actions of newer heparins and antithrombotic drugs. Studies suggest that the pathophysiologic responses after a thrombogenic trigger in the primate model are close to humans, and drug modulation of these may provide relevant clinical information. This model provides the most similar preclinical model to study the actions of heparin fractions.     

Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage

We cloned two distinct cDNAs for enteropeptidase (EP) from the intestine of the medaka, Oryzias latipes, which is a small freshwater teleost. The mRNAs code for EP-1 (1,036 residues) and EP-2 (1,043 residues), both of which have a unique, conserved domain structure of the N-terminal heavy chain and C-terminal catalytic serine protease light chain. When compared with mammalian EP serine proteases, the medaka enzyme exhibited extremely low amidolytic activity for small synthetic peptide substrates. Twelve mutated forms of the medaka EP protease were produced by site-directed mutagenesis. Among them, one mutant protease, E173A, was found to have considerably reduced nonspecific hydrolytic activities both for synthetic and protein substrates without serious reduction of its Asp-Asp-Asp-Asp-Lys (D(4)K)-cleavage activity. For the cleavage of fusion proteins containing a D(4)K-cleavage site, the medaka EP proteases were shown to have advantages over their mammalian counterparts. Based on our present data, we propose that the E173A mutant is the most appropriate protease to specifically cleave proteins containing the D(4)K cleavage sequence.     

寄生线虫丝氨酸蛋白酶抑制剂研究进展

丝氨酸蛋白酶抑制剂在寄生线虫抵抗宿主蛋白酶对虫体损伤、调控炎症与免疫应答以及获得营养中发挥重要作用,部分抑制剂还参与虫体的发育及生殖.该文对寄生线虫中的丝氨酸蛋白酶抑制剂研究进展作一综述.     

A major serine protease in rat skeletal muscle: Evidence for its mast cell origin

The physical, chemical, and immunologic properties of a protease from rat skeletal muscle, proposed to function in the degradation of certain intracellular enzymes, are identical to those of a chymotrypsin-like serine protease isolated from peritoneal mast cells. The results of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea indicate that the two rat proteases have identical mobilities corresponding to a molecular weight of 26,000. The relative amino acid compositions of the proteases are nearly identical. Immunodiffusion tests for crossreaction between the muscle protease and antisera directed toward mast cell protease indicate that the former is immunologically identical to mast cell protease. The first 35 amino-terminal residues of the two enzymes are identical and indicate homology of these proteins to other mammalian serine proteases. The sequence analysis of the protease from muscle was extended for an additional 16 positions, and comparison of this amino-terminal sequence with that of a similar enzyme from small intestine showed approximately 75% sequence identity. In contrast, only 40% of the residues in this region of bovine chymotrypsin A were found at corresponding loci in rat muscle protease. It is concluded that the protease from muscle or mast cells is closely related to the enzyme from small intestine which recently was localized in the "atypical" mast cells of gut mucosa [Woodbury, R. G., Gruzenski, G. M. & Lagunoff, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2785-2789].     

斯氏按蚊丝氨酸蛋白酶基因克隆及其与约氏疟原虫卵囊黑化关系的研究

目的克隆斯氏按蚊丝氨酸蛋白酶基因,分析该分子与约氏疟原虫卵囊黑化关系。方法根据其它昆虫丝氨酸蛋白酶的保守氨基酸序列设计简并引物,以斯氏按蚊全蚊cDNA为模板,进行RT-PCR扩增,PCR产物纯化,克隆入pUCm(T载体,测定插入片段序列,对序列进行BLAST检索和鉴定,根据序列设计相应基因的特异引物,然后分别从血淋巴细胞或中肠扩增目的基因,并进行不同食源条件下丝氨酸蛋白酶基因的半定量、原位核酸分子杂交定位与约氏疟原虫卵囊黑化关系的分析。结果获得了2种斯氏按蚊血细胞丝氨酸蛋白酶cDNA部分序列,即AsSP1(448bp)和AsSP2(472bp),分别与冈比亚按蚊SP2A和大劣按蚊SP2基因很相似,其编码的氨基酸序列均包含保守的丝氨酸蛋白酶催化功能域。在中肠和血淋巴内也获得相同的目的基因片段,半定量分析显示约氏疟原虫感染或诱导卵囊黑化呈现之前的表达明显增强,原位核酸分子杂交技术也进一步证实血细胞有AsSP1和AsSP2 mRNA表达。结论AsSP1和AsSP2很可能与疟原虫感染或约氏疟原虫卵囊黑化相关,推测可能是斯氏按蚊的前酚氧化酶的活化酶。