Plasminogen activators: general aspects and recent developments

Considerable interest in plasminogen activators as human thrombolytic drugs has stimulated rapid biotechnologic progresses. These enzymes have been classified in two immunochemically distinct groups: "urokinase-like" activators or u-PA which do not interact with fibrin and "tissue activator-like" activators or t-PA which interact with fibrin. Plasminogen activators are widely distributed in normal and malignant tissues and they are implicated in various physiological and pathological processes. They maintain the functional integrity of the vascular system and their presence may be of importance in tissue remodeling and cell migration. Urokinase and streptokinase are used in human thrombolytic therapy. However, the properties displayed by t-PA suggest that this enzyme may be a superior fibrinolytic agent. The primary structures of urokinase and t-PA are known; both enzymes have been synthesized by DNA technology. In order to produce t-PA in large quantities by gene cloning, intensive studies are conducted by pharmaceutical industries. Clinical trials using t-PA for dissolving thrombi in coronary heart disease, strokes and pulmonary embolism are in progress. This review presents the molecular and structural properties of plasminogen activators, as well as related physiological, pathological and therapeutic aspects.     

Visible fibrinolysis by endothelial cells: Effect of vitamins and sterols

We have succeeded in corroborating the enhancing effect of vitamin A, vitamin C, sitosterol and fucosterol on the fibrinolytic activity of endothelial cells. The assay system consisted of anin situ dissolution of a fibrin layer coated onto a culture dish, over which endothelial cells were grown in a culture medium containing 10 % serum. The dissolution was enhanced by the addition of these vitamins and phytosterols to the culture medium.To whom correspondence should be addressed.     

Fibrinolysis: Its initiation and regulation

Fibrinolytic system is one of the major proteolytic pathways in vivo and primarily responsible for dissolution of thrombi. Two enzymes are primarily involved in this proteolytic system; plasminogen activator (PA) and plasmin. Plasmin is formed by a limited proteolysis of plasminogen by PA, which is mainly synthesized by and secreted from vascular endothelial cells. This proteolytic process proceeds physiologically only on the surface of fibrin. Thus, initiation and progression of the fibrinolytic process depend on the function of endothelial cells and fibrin formation. Endothelial cells may also synthesize and excrete PA inhibitor (PAI) which inhibits immediately, PA once released. The rates of synthesis and excretion of PA and PAI by endothelial cells are regulated by various factors. Among them, thrombin stimulates the release of PA whereas activated protein C may decrease the release of PAI. Thus, both enzymes enhance fibrinolytic potential. PA which has escaped from inhibition by PAI binds to fibrin. ₂-Plasmin inhibitor (₂PI) inhibits the binding of plasminogen to fibrin, thereby suppressing this fibrin-associated plasminogen activation. A part of ₂PI is cross-linked to fibrin by activated factor XIII when fibrin is formed, and the ₂PI thus cross-linked to fibrin inhibits in situ plasmin formed on fibrin. Thus, ₂PI as well as PAI plays a central role in inhibition of fibrinolysis.     

Structure and function of human tissue-type plasminogen activator (t-PA)

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.     

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.     

嗜麦芽寡养单胞菌胞外蛋白酶功能研究进展

嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)是广泛分布于自然界的革兰氏阴性杆菌。作为一种新型、与高死亡率相关的条件致病菌,嗜麦芽寡养单胞菌能够导致人类或其他生物感染多种疾病。近年来,越来越多的研究结果显示来自于细菌的胞外蛋白酶是导致宿主发病的关键蛋白质。因此,探究嗜麦芽寡养单胞菌胞外蛋白酶的组成成分和功能将不仅有助于阐明其致病机制,更为今后以其为靶点进行临床治疗奠定基础。本文试图对嗜麦芽寡养单胞菌胞外蛋白酶的性质、功能及其应用进行归纳总结。     

DIURNAL VARIATION OF HAEMOSTATIC RESPONSE TO EXERCISE IN YOUNG SEDENTARY MALES

The aim of this study was to evaluate diurnal variations in the haemostatic response to submaximal exercise performed by young, sedentary men. Fifteen healthy young sedentary males aged 25.6 ± 1.34 (mean ± SD) years performed two exercise sessions, morning and evening, at 70% of maximal oxygen consumption (V.O₂max) on a cycle ergometer for 30 min. Platelet count (PC), activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen, tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) activity were measured as dependent variables. Exercise produced significant increases in PC and fibrinogen for both sessions (P ≤ 0.05), which returned to the resting values after recovery only in the evening session. APTT and PT shortened immediately after exercise, which remained after recovery for both sessions (P ≤ 0.01). Exercise presented significant increases in tPA activity (P ≤ 0.001), which returned to the baseline after recovery in both exercises. PAI-1 activity was significantly higher during the morning than evening (P ≤ 0.05), but no longer demonstrated exercise-related changes. It was found that exercise caused activation of both coagulation and fibrinolysis processes, partly related to the time of the day when the exercise was performed.     

Inhibitory Potency of Erythrina variegata Proteinase Inhibitors toward Serine Proteinases in the Blood Coagulation and Fibrinolytic Systems

The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman–Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIa inhibited plasma kallikrein. Neither ETIa nor ETIb exhibited any inhibitory activity toward β-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence.     

CPR-Total (TAFI and activated TAFI) levels in plasma/serum of hemophiliacs

Arginine carboxypeptidase (CPR) is a single-chain plasma protein generated during coagulation from a precursor (proCPR). proCPR is the same molecule as thrombin activable fibrinolysis inhibitor (TAFI), which retards fibrin clot lysis in vitro and most likely modulates fibrinolysis in vivo. In this study, the amount of CPR-total, which includes proCPR (TAFI) and CPR (activated TAFI), in hemophiliac patients was evaluated using a newly developed enzyme linked immunosorbent assay (ELISA). The amount of CPR-total in plasma or serum of most of the hemophiliac patients was in the range of healthy individuals. There was no significant difference in hemophiliac patients with or without HIV-1 infection. However, two out of the 74 hemophiliac patients showed a significantly high level. The upregulation of CPR-total might contribute to compensate for inefficient coagulation in some hemophiliac individuals.     

改良乳腺癌根治术后切口感染的病原学特征、影响因素及其凝血纤溶功能研究

摘要 目的：探讨改良乳腺癌根治术后切口感染的病原学特征、影响因素及其对凝血纤溶功能的影响。方法：选取我院于2016年6月~2020年9月期间收治的390例行改良乳腺癌根治术的乳腺癌患者,分析改良乳腺癌根治术后切口感染的病原学特征、影响因素及术后切口感染对凝血纤溶功能的影响。结果：390例行改良乳腺癌根治术的乳腺癌患者,术后发生切口感染28例,术后切口感染率为7.18%（28/390）,将未发生术后切口感染的患者纳为未感染组（n=362）,发生的纳为感染组（n=28）。28例发生感染的患者共分离培养病原菌36株,其中革兰阳性菌14株,占比38.89%（14/36）,以金黄色葡萄球菌、粪肠球菌为主。革兰阴性菌21株,占比58.33%（21/36）,以大肠埃希菌、铜绿假单胞菌为主。改良根治术后乳腺癌患者切口感染的影响因素包括手术时间、术后住院时间、合并基础疾病、引流时间、年龄、白蛋白（P<0.05）。多因素Logistic回归分析发现：合并基础疾病、年龄≥60岁、白蛋白＜35 g/L、手术时间≥120 min均是改良乳腺癌根治术后切口感染的影响因素（P＜0.05）。两组术后30 d凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血酶时间（TT）升高,且未感染组高于感染组（P<0.05）,纤维蛋白原（FIB）降低,且未感染组低于感染组（P<0.05）。结论：改良乳腺癌根治术后切口感染较为常见,致病菌以革兰阴性菌为主,年龄、合并基础疾病、白蛋白、手术时间均是其影响因素,同时术后切口感染的发生可影响凝血纤溶功能的恢复,临床医生应积极采取措施预防术后切口感染的发生,从而保证手术治疗效果。