Mechanism Of Thrombin Research Articles

Thrombin Improves Diabetic Wound Healing by ERK-Dependent and Independent Smad2/3 Linker Region Phosphorylation.

Impaired wound healing is one of the most noteworthy features and troublesome complications of diabetes mellitus, which arouses a rising global health concern without potent remedies. Thrombin is the major hemostatic agent applied at wound healing initiation and recently gained therapeutic credits in later phases. However, a rare investigation achieved prolonged use of thrombin and probed the detailed mechanism. The objective of this study is to investigate the effects and mechanism of thrombin on diabetic skin wound healing. The effect of thrombin on fibroblast proliferation, α-SMA, and Collagen I expression was firstly studied in vitro by Cell Counting Kit 8 (CCK8) and western blotting. Then, the specific phosphorylation site of SMAD2/3 and their ERK1/2 dependence during thrombin treatment were assessed by western blotting for mechanism exploration. After that, full-thickness wound defects were established in diabetic male SD rats and treated with thrombin in the presence or absence of PD98059 to observe the in vivo effects of thrombin and to confirm its ERK dependence. We found that thrombin promoted fibroblast proliferation and their α-SMA and Collagen I production. Mechanistically, thrombin induced phosphorylation of Smad2 linker region (Ser245/250/255) through ERK1/2 phosphorylation but promoted phosphorylation of Smad3 linker region (Ser204) independent of ERK1/2. Histological results showed that thrombin facilitated wound healing by promoting α-SMA and Collagen I expression, which was not abolished by inhibiting ERK phosphorylation. Collectively, this study validated the therapeutic efficacy of thrombin on diabetic wound healing and identified both ERK-dependent and -independent Smad2/3 linker region phosphorylation as the essential signaling events in this process.

Thrombin Aggravates Hypoxia/Reoxygenation Injury of Cardiomyocytes by Activating an Autophagy Pathway-Mediated by SIRT1.

BackgroundAcute myocardial infarction is the leading cause of mortality among adults worldwide. The present study aimed to investigate the role and mechanism of thrombin and SIRT1 in hypoxia/reoxygenation (H/R) injury.Material/MethodsH9c2 cardiomyocytes were used to create an H/R model to simulate in vivo ischemia/reperfusion injury. The MTT assay was used to measure cell viability, qRT-PCR was used to detect the level of SIRT1, thrombin, and PAR-1, and western blot analysis was conducted for evaluation of thrombin, PAR-1, SIRT1, LC3I, LC3II, and Beclin1. ELISA was applied for determination of IL-1b, IL-6, TNF-a, MMP-9, and ICAM-1. After the establishment of the H/R model, superoxide dismutase (SOD) activity was evaluated by the xanthine oxidase method, malondialdehyde content was detected by thiobarbituric acid assay, and reactive oxygen species generation was measured by CM-H2DCFDA.ResultsThe findings showed that thrombin enhanced inflammatory factor secretion and oxidative stress but inhibited cell viability in H/R-injured cardiomyocytes. We also observed that thrombin promoted autophagy in H/R-injured cardiomyocytes. In addition, thrombin enhanced the upregulation of SIRT1 expression by H/R. However, it was found that inhibition of SIRT1 could suppress the effect of thrombin on inflammatory factor secretion, oxidative stress, and cell viability. Moreover, downregulation of SIRT1 suppressed the inhibitory effect of thrombin on autophagy in H/R injury.ConclusionsThrombin aggravates H/R injury of cardiomyocytes by activating an autophagy pathway mediated by SIRT1. These findings might provide a potential target therapy for the treatment of ischemia/reperfusion injury in future clinical work.

Heparin-like tubings: III. Kinetics and mechanism of thrombin, antithrombin III and thrombin-antithrombin complex adsorption under controlled-flow conditions

In previous papers, we described treated tubular materials which exhibit an heparin-like antithrombic activity under dynamic conditions. In order to ascertain the heparin-like mechanism of this activity, we have studied the interactions of thrombin, antithrombin III and thrombin-antithrombin III complex with the inner face of these treated tubings under controlled-flow conditions. Moreover, the kinetics of the adsorption of thrombin were studied at different flow rates to establish the rate-determining step.

Studies on the mechanism of thrombin. Interaction with fibrin.

Fibrin monomer Sepharose was used to investigate the interactions of thrombin with fibrin. Thrombin binding was found to be reversible and saturable and to depend on the thrombin: fibrin ratio. Scatchard analysis indicated a single class of binding sites with K alpha = 4.9 X 10(5) M-1. Ca2+ ions caused rapid desorption and elution of thrombin from fibrin monomer, and the Ca2+ concentration needed for maximal desorption depended on the fibrin:thrombin ratio. Mg2+, Mn2+, and Sr2+ also released thrombin from fibrin monomer but not as efficiently as Ca2+. These results indicate that divalent metal ions induce a physical change in fibrin monomer which results in desorption of thrombin. Thrombin binding to fibrin in a gel was compared to binding to fibrin monomer. These studies showed that as fibrin monomers polymerize to form the gel network, thrombin is released. Under static conditions the released thrombin remains associated with the gel because diffusion is limited by the gel. However, the thrombin can be readily removed when buffer is allowed to flow through the gel. These results lead to the possibility that thrombin binding to fibrin monomer and its subsequent release, either by Ca2+ or by polymerization, may have important consequences for regulating the effective thrombin concentration in vivo.

Mathematical model of the activation of prothrombin by factor Xa and factory Vt.

A mathematical model of prothrombin activation is being proposed which includes the feedback mechanism of thrombin and the alteration of factor V by thrombin. This model is in good agreement with experimental data for the dependence of the rate of thrombin formation on the concentrations of factors V and Xa. In particular, it correctly predicts the existence and location of a maximum in both of these cases.

Fundamentals of blood clotting.

This review covers the interval between July 1, 1949, and June 30, 1951. Obviously, space limitations preclude a complete citation of every paper. The older literature is cited when required for clarity or readability. For col­ lateral reading, the excellent review of Astrup (1) is recommended. Like­ wise of particular interest are the series of the Transactions of the Confer­ ences on Blood Clotting and Allied Problems (2, 3, 4,5), which include un­ embellished discussions of many subjects covered in this review. The blood clotting literature can be divided into three parts: funda­ mental aspects, anticoagulation therapy, and hemostasis. The fundamental aspects are emphasized in this review. The book by Marple & Wright (6) is available for those interested in anticoagulation therapy. A recent book by Quick (7) covers the problem of hemostasis. A review by Koller (8) sum­ marizes some of the papers not cited in this article. Prior to 1943, many investigators believed that all essential factors re­ quired for blood clotting were included in the classical theory which held that the formation of thrombin results from the interaction of thromboplas­ tin, calcium, and prothrombin. It is now clearly demonstrated that at least one or two more factors are involved in the mechanism of thrombin forma­ tion. One of these factors is variously termed thrombogen (9, 10), prothrom­ bin A or labile factor (11, 12), plasmakinin (13, 14), prothrombin accelel'ator (15), factor V (16) (i.e., the fifth factor in blood clotting), proaccelerin (17), accelerin (1) , and accelerator globulin (IS}. The consensus of most workers is that all of the previously named factors are one and the same. In addition, a number of investigators believe that still other factors exist. For descriptive purposes, the new factors are dis­ tinguishable from the classical ones by the qualifying term accessory. This term does not imply that they possess merely a supplementary action to the classical factors; indeed, they may be as necessary as prothrombin.