Reduction of thrombus size in murine models of thrombosis following administration of recombinant α1-proteinase inhibitor mutant proteins

The variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1-75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R. Recombinant serpins were purified from Escherichia coli by nickel chelate and ion exchange affinity chromatography, and administered to mice intravenously. HAPI RCL5 reduced incorporation of radiolabelled fibrin(ogen) into thrombi in the ferric chloride-injured vena cava in a dose-dependent manner; HAPI M358R was less effective and α1-PI M358R was without effect. In a model of murine endotoxaemia, HAPI RCL5 was more effective than α1-PI M358R in reducing radiolabelled fibrin(ogen) deposition in heart and kidneys; immunohistochemistry of tissue sections showed lesser staining with anti-fibrin(ogen) antibodies with both treatments. In the ferric chloride-injured murine carotid artery, administration of both recombinant serpins was equally effective in lengthening the vessel's time to occlusion. Our results show that the antithrombotic efficacy of the recombinant serpins correlates with their potency as thrombin inhibitors, since HAPI RCL5 inhibits thrombin, but not factors Xa, XIa, XIIa, or neutrophil elastase, more rapidly than α1-PI M358R.     

The appended tail region of heparin cofactor II and additional reactive centre loop mutations combine to increase the reactivity and specificity of alpha1-proteinase inhibitor M358R for thrombin

Natural inhibitors of coagulation or inflammation such as the serpins antithrombin (AT), heparin cofactor II (HCII), and alpha(1)-proteinase inhibitor (alpha(1)-PI) can be overwhelmed in thrombosis and/or sepsis. The reactive centre (P1-P1') variant alpha(1)-PI M358R inhibits not only procoagulant thrombin but also anticoagulant activated protein C (APC). We previously described HAPI M358R, comprising a fusion of HCII residues 1-75 to the N-terminus of a(1)-PI M358R that yielded increased anti-thrombin, but not anti-APC activity. We hypothesized that further alterations to the HAPI M358R reactive centre loop would yield additional refinements in specificity. The reactions with thrombin or APC of recombinant alpha(1)-PI M358R variants with or without the HCII extension were characterized electrophoretically and kinetically. Their extension of clotting times and inhibition of fibrin-bound thrombin were measured, and the survival of HAPI M358R in mice was determined. Replacing the P7-P3 and P2' residues of HAPI M358R with AT residues reduced APC inhibition rates by 140-fold, but those of thrombin less than two-fold;substituting the P16-P2 and P2'-P3' residues of HAPI M358R with HCII residues reduced APC inhibition rates by 180-fold, but those of thrombin 10.5-fold. Fused variants extended thrombin clotting times more effectively than unfused inhibitors, were at least as effective at inhibiting clot-bound thrombin, and remained intact in the murine circulation. The combination of modifications inside and outside the RCL resulted in a 1,360-fold increase in selectivity of HAPI M358R (AT P7-P3/P2') for thrombin versus APC relative to alpha(1)-PI M358R. Our results predict that this protein may be effective in limiting thrombosis in vivo.     

Phospholipid-bound tissue factor modulates both thrombin generation and APC-mediated factor Va inactivation

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.     

Tissue factor over-expression by human pancreatic cancer cells BXPC3 is related to higher prothrombotic potential as compared to breast cancer cells MCF7

Cancer histology influences the risk of venous thromboembolism and tissue factor (TF) is the key molecule in cancer-induced hypercoagulability. We investigated the relation between TF expression by pancreatic and breast cancer cells (BXPC3 and MCF7 respectively) and their capacity to trigger in vitro thrombin generation in normal human plasma. Flow cytometry and Western blot analysis for TF expression were performed using murine IgG1 monoclonal antibody against human TF. Real-time PCR for TFmRNA was also performed. Activity of TF expressed by cancer cells was measured with a specific chromogenic assay. Thrombin generation in PPP was assessed using calibrated automated thrombogram. Cancer cells were added to platelet poor plasma from healthy volunteers. In separate experiments cells were incubated with the anti-TF antibody at concentration that completely neutralized the activity of recombinant human TF on thrombin generation. BXPC3 cells expressed significantly higher amounts of functional TF as compared to MCF7 cells. Incubation of BXPC3 and MCF7 cells with PPP resulted in acceleration of the initiation phase of thrombin generation. BXPC3 cells manifested higher procoagulant potential than MCF7 cells. The incubation of BXPC3 or MCF7 cells with the anti-TF monoclonal antibody which resulted in reversal of their effect on thrombin generation.The present study establishes a link between the amount of TF expressed by cancer cells with their procoagulant activity. Both studied types of cancer cells trigger thrombin generation but they have different procoagulant potential. The procoagulant activity of BXPC3 and MCF7 cells is related to the amount of TF expressed. Kinetic parameters of thrombogram are the most relevant for the detection of the TF-dependent procoagulant activity of cancer cells. TF expression is one of the mechanisms by which cancer cells manifest their procoagulant potential but it is not the unique one. The present experimental model will allow the characterization the procoagulant fingerprint of cell lines from the same or different histological types of cancer.     

Atorvastatin reduces thrombin generation and expression of tissue factor, P-selectin and GPIIIa on platelet-derived microparticles in patients with peripheral arterial occlusive disease

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD61), P-selectin (CD62P), tissue factor (TF, CD142) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of prothrombin fragment 1+2 in plasma (F1+2) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.001 for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.001) and by measurements of F1+2 (p<0.01). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.     

Enhanced effect of inhibition of thrombin on endothelium in murine endotoxaemia: Specific inhibition of thrombocytopenia

Introduction

In systemic endotoxaemia, bacterial lipopolysaccharide causes the rapid expression of tissue factor (TF) and disseminated intravascular coagulation and in animal models, anticoagulants limit pathology and promote survival. Recent studies have emphasised the importance of TF expressed by mononuclear cells for initiating thrombin generation during endotoxaemia and suggested that endothelial cell TF is of little relevance. However, the precise importance of endothelium for intravascular thrombin generation has not been established. In this study, we compared the effect of equivalent levels of hirudin tethered to either endothelium or platelets and monocytes.

Materials and Methods

CD31-Hir-Tg mice express a vesicle-targeted, membrane-tethered hirudin fusion protein on endothelium, platelets and monocytes. Bone marrow chimeras between these mice and C57BL/6 were generated The level of intravascular hirudin expressed during endotoxaemia was quantified by inhibition studies using an anti-hirudin antibody and reference to the circulating thrombin anti-thrombin complexes generated in control mice given soluble hirudin.

Results and Conclusions

Antibody inhibition studies indicated that individual chimeras expressed similar levels of hirudin fusion protein on endothelium alone as on platelets and leukocytes combined and accordingly, the levels of thrombin anti-thrombin complexes and fibrinogen in each chimera were similar, indicating equivalent inhibition of thrombin generation. However, mice with hirudin on endothelium alone developed significantly less thrombocytopenia. These results suggest a hitherto unrecognized role of endothelium in thrombin-dependent platelet sequestration during endotoxaemia. The data have implications for the development of therapeutic strategies based on targeted anticoagulation to limit disseminated intravascular coagulation.     

Activated protein C generation is greatly decreased in plasma from newborns compared to adults in the presence or absence of endothelium

Activated protein C (APC) generation strongly affects sepsis and thrombosis by inhibition of thrombin generation. However, it is unclear if there are age-related differences in effectiveness of protein C (PC). We studied age effects on plasma APC generation +/- endothelium. Defibrinated (Ancrod) plasma (from adults or newborns (umbilical cord)) was recalcified with buffer containing tissue factor +/- thrombomodulin (TM) on either plastic or endothelium (HUVEC) at 37 degrees C. Timed subsamples of reaction mixture were taken into either heparin-EDTA or FFRCMK-EDTA solutions and analyzed for APC-PC inhibitor (APC-PCI) or APC-alpha1 antitrypsin (APC-alpha1 AT) by ELISAs. Since heparin converts free APC to APC-PCI, the difference in APC-PCI measured in heparin-EDTA and FFRCMK-EDTA samples was equal to free active APC. APC-alpha2 macroglobulin (APC-alpha2M) was measured as remaining chromogenic activity in heparin-EDTA. Free APC, APC-PCI and APC-alpha1 AT were decreased in newborn compared to adult plasma on plastic. However, APC-alpha2M made up a larger fraction of inhibitor complexes in new-born plasma. On endothelium, significantly more APC, APC-PCI and APC-alpha1AT were generated in either plasma compared to that on plastic with excess added TM. APC, APC-PCI and APC-alpha1AT were also reduced and total APC-alpha2M increased in newborn plasma on HUVEC. Addition of PC to newborn plasma gave APC generation similar to adult plasma. Thus, free APC, APC-PCI and APC-alpha1AT generation is reduced in newborn compared to adult plasma with or without endothelium, likely due to reduced plasma PC levels. Endothelium enhances APC generation, regardless of plasma type, possibly because of cell surface factors such as TM, phospholipid and endothelial PC receptor.     

Acute release of tissue factor pathway inhibitor after in vivo thrombin generation in baboons

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.     

Anticoagulant effect of inositol hexasulfate as measurable by clotting times of fibrinogen and recalcified plasma

Thrombin-clotting time of fibrinogen was delayed by inositol hexasulfate (IHS). Clotting time of the recalcified plasma was also delayed by IHS. Moreover, when IHS was intravenously injected into anesthetized male rabbits (0.11 mmoles/kg), it delayed clotting time of the recalcified plasma. IHS prevented generations of factor Xa and thrombin in the recalcified plasma. It is probable that IHS inhibits the clot-formation of the recalcified plasma in competition with calcium ions.     

Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma

The thrombin generation test is used to study coagulation in patients with haemorrhagic diseases or with high thrombotic risk. To our knowledge, this is the first study investigating the relative influence of coagulation factors on thrombin generation in plasma. The aim was to investigate the influence of coagulant factors, anticoagulant factors, and tissue factor (TF) on three parameters: endogenous thrombin potential (ETP), peak thrombin concentration, and lag time for the appearance of thrombin. At a low TF concentration, all factors except factor XI influenced thrombin generation. At a high TF concentration, only the factors of the extrinsic pathway exerted an influence. ETP and peak thrombin were linearly correlated to factor II concentration. Factor V and factor VII effects increased hyperbolically with factor concentration. The influence of factor X on thrombin generation depended on TF concentration. In the absence of factor VIII and factor IX, ETP fell to 60-70% of the normal when peak thrombin fell to 25-30% of the normal. Fibrinogen concentration influenced ETP and peak thrombin and decreasing fibrinogen levels shortened the lag time. As expected, decreasing antithrombin concentration caused dramatic increases in thrombin generation. Protein S prolonged the lag time, especially at a low TF concentration. No effect of protein C was observed, likely due to the absence of thrombomodulin. The thrombin generation test was more sensitive to factor deficiencies at low than at high TF concentration. ETP was not the most critical parameter for studying coagulation factor deficiencies. Instead, peak thrombin was the most sensitive parameter.     

Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Background

The calibrated automated thrombogram (CAT) assay measures thrombin generation in plasma.

Objective

Use the CAT assay to detect endogenous tissue factor (TF) in recalcified platelet-rich plasma (PRP) and platelet-free plasma (PFP).

Methods

Blood from healthy volunteers was collected into citrate and incubated at 37 °C with or without lipopolysaccharide (LPS) for 5 hours. PRP and PFP were prepared and clotting was initiated by recalcification. Thrombin generation was measured using the CAT assay.

Results

The lag time (LT) was significantly shortened in PRP prepared from LPS-treated blood compared with untreated blood (10 ± 3 min versus 20 ± 6 min), and this change was reversed by the addition of inactivated human factor VIIa. LPS stimulation did not change the peak thrombin. Similar results were observed in PFP (21 ± 4 min versus 35 ± 5 min). LPS stimulation also significantly reduced the LT of PRP and PFP derived from blood containing citrate and a factor XIIa inhibitor. Finally, a low concentration of exogenous TF shortened the LT of PFP prepared from unstimulated, citrated blood without affecting the peak thrombin.

Conclusion

Changes in LT in the CAT assay can be used to monitor levels of endogenous TF in citrated plasma.     

Alternatively spliced human tissue factor (asHTF) is not pro-coagulant

It has been proposed that alternatively-spliced human tissue factor (asHTF) is pro-coagulant. We have evaluated the function of asHTF in a mammalian expression system. Full-length human tissue factor (HTF) and asHTF were cloned from smooth muscle cells and over-expressed in HEK293 cells. As expected, a marked pro-coagulant activity (FX activation, thrombin generation) was observed on the surface, in lysates, and on microparticles from HTF transfected cells. In contrast, no pro-coagulant activity of asHTF was observed.     

Platelet tissue factor pathway inhibitor modulates intravascular coagulation

Tissue factor pathway inhibitor (TFPI) is produced by megakaryocytes and is found internally within quiescent platelets but is not in α-granules. It is released in soluble form and expressed on the surface of platelets that are dual activated with thrombin plus collagen. Platelet TFPI is exclusively TFPIα, the most evolutionarily conserved TFPI isoform. It appears to be physiologically active as an inhibitor of tissue factor (TF) initiated FXa generation in vitro, and acts locally to dampen clot growth in a murine vascular injury model. These data suggest that platelet TFPI plays an important role in modulating TF activity within a growing clot thereby preventing formation of an occlusive clot.     

Antithrombin I. Inhibition of thrombin generation in plasma by fibrin formation

Thrombin substrate binding is mediated through fibrinogen recognition "exosite 1" in thrombin, resulting in fibrinopeptide cleavage to form fibrin. In addition, thrombin exhibits "non-substrate" binding to fibrin, an activity termed "Antithrombin I". Antithrombin I (AT-I) is characterized by two classes of thrombin binding sites, the first of "low affinity" in the fibrin E domain, and the other of high affinity, that is situated between C-terminal residues 414 and 427 of a variant gamma chain termed gamma'(1-427L), Plasma fibrinogen molecules containing gamma' chains ("fibrinogen 2") are virtually all heterodimers containing one gamma(A) chain (platelet-binding) and one gamma' chain. The remaining fibrinogen (approximately 85%) is homodimeric, lacks high affinity thrombin-binding potential, and is termed " fibrinogen 1" (gamma(A)/gamma(A)). Thrombin generation in recalcified fibrinogen-depleted or congenital afibrinogenemic plasma is increased. Repletion with fibrinogen 1 has a modest effect in normalizing thrombin generation, whereas repletion with fibrinogen 2 (gamma(A)/gamma') has a more marked effect. A post-translational gamma' chain derivative, gamma'(1-423P), accounts for 3%-34% of the gamma' chain population, lacks thrombin binding potential, and arises by proteolytic processing at the expense of gamma' (1-427L) chains. Little is known about its effect on plasma AT-I activity under normal or pathological circumstances. In summary, fibrin formation (Antithrombin I) inhibits thrombin generation in clotting blood by sequestering thrombin, and "high-affinity" thrombin-binding (i.e., via gamma' chains) plays a dominant role in this process. AT-1 should be considered when assessing the pathogenesis of thromboembolic disease.     

Effects of EACA on thrombin generation as measured by the chromagen S2238

In the presence of epsilon aminocaproic acid (EACA) thrombin generation in recalcified platelet rich plasma (PRP) was markedly stimulated, as measured by the cleavage of the synthetic substrate S2238. However, thrombin activity remaining after 30 minutes incubation was reduced when compared with control values. The residual activity was shown to be hirudin insensitive and to be associated with a species of higher molecular weight than free thrombin. These results suggested an inhibition of thrombin binding to the antithrombin, alpha 2-macroglobulin (₂M). Preincubation of PRP with EACA reduced the concentration at which EACA elicited its dual effects. Similar results were obtained with the ₂M inhibitor, hydrazine: These experiments indicated that ₂M may play a more important role in regulating thrombin generation than has been previously recognized.     

Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells

Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997;19(October (4)):735-41], SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (1 Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T. Eur J Neurosci 2001;13(June (12)):2314-18] were expressed, imaged and voltage clamped in HEK 293 cells and in dissociated hippocampal neurons. We were unable to detect a signal in response to changes in membrane potential after averaging16 trials with any of the three constructs. Using the hydrophobic voltage sensitive dye, di8-ANEPPS, as a surface marker, confocal analyses demonstrated poor plasma membrane expression for Flare, SPARC and VSFP-1 in both HEK 293 cells and dissociated hippocampal neurons. Almost all of the expressed FP-voltage sensors reside in internal membranes in both cell types. This internal expression generates a background fluorescence that increases the noise in the optical measurement.     

Tissue factor pathway inhibitor on circulating microparticles in acute myocardial infarction

In acute myocardial infarction (AMI), increased Tissue Factor (TF) expression on circulating monocytes and microparticles (MP) may contribute to thrombotic events. Because surfacebound Tissue Factor Pathway Inhibitor-1 (TFPI) inhibits TF activity on monocytes and endothelial cells decreased TFPI expression may reinforce the procoagulant activity of circulating MP. Aim of the study was to analyze TFPI expression and TF activity after stenting and thrombolysis inAMI. Thirty-nine patients of a randomized study comparing intravenous thrombolysis (n=19) and stenting (n=20) were included. Before and after therapy blood samples for analysis of MPs, TF antigen and activity, prothrombin fragment F1+2 and D-dimer were obtained. TFPI expression on TF positive MPs was decreased after thrombolysis but not after stenting. In contrast, TF plasma levels and TF positive MP remained unchanged in both treatment groups. After thrombolysis increased D-dimer and F1+2 plasma concentrations indicated activation of fibrinolysis and coagulation. Significance of MPTFPI for inhibition of TF activity was measured using inhibitory TFPI antibodies. Membrane-associated TFPI inhibited TF activity on circulating MPs. After thrombolysis inhibition of TF activity by TFPI was decreased as compared to stenting. Correlation of circulating TF with F1+2 only after thrombolysis, suggests a role for TF-induced activation of coagulation after thrombolysis. Enhanced TF activity on circulating MPs in AMI is inhibited by endogenous surface-boundTFPI. After thrombolysis but not after stenting MPTFPI is degraded and may induce thrombin generation due to unopposed tissue factor activity. Anti-TF therapies during thrombolysis may reduce thrombin generation in AMI.     

Tissue factor expression in blood cells

The popular concept of TF serving predominantly as a hemostatic envelope encapsulating the vascular bed, has recently been challenged by the observation that blood of healthy individuals may form TF-induced thrombus under conditions entailing shear stress and activated platelets, corroborating the notion of blood borne TF. Accordingly, small amounts of TF activity is detected in calcium ionophore-stimulated monocytes, whereas it is questionable whether neutrophils and eosinophils express TF. Still there are contradicting reports on TF synthesis and expression in activated platelets, but when using a very sensitive and specific assay for TF activity measurements, we fail to detect TF activity associated with platelets activated with various agonists. However, activated platelets may play a role in decrypting monocyte TF activity in a process entailing transfer of TF to activated platelets in a P-seelctin –PSGL-1 reaction whereby inactive TF (encrypted) becomes active through the availability of clusters of phosphatidylserine. Microparticles from plasma of healthy subjects possess weak TF-like activity which is not inactivated by anti-TF antibody. Endothelial cells are well documented to synthesize TF by several agonists in vitro. In contrast, there is little evidence that these cells are capable of synthesizing TF in vivo, and a recent report fails to show that TF on the endothelium may play any role in thrombin generation in a murine endotoxemia model. It may be concluded that monocytes are the only blood cells that synthesize and express TF and which may be the only source for TF-induced thrombosis when the endothelium is intact.     

突触蛋白I片段促进α-突触核蛋白的聚集

目的 探讨突触蛋白I(Synapsin I,Syn I)被剪切后形成的C83片段对α-突触核蛋白聚集的影响。方法 在稳定表达α-突触核蛋白的HEK293细胞、小鼠原代神经元以及Tau P301S转基因小鼠体内分别过表达Syn I全长及其C83片段,通过细胞免疫荧光染色、蛋白免疫印迹技术以及免疫组织化学染色的方法观察Syn I全长及其C83片段对α-突触核蛋白磷酸化及聚集的影响。结果 Syn I C83片段促进HEK293细胞中α-突触核蛋白的磷酸化、泛素化以及聚集,并诱导小鼠原代神经元以及Tau P301S转基因小鼠体内α-突触核蛋白的磷酸化和聚集。结论 在细胞和动物模型中Syn I C83片段均可以促进α-突触核蛋白的聚集,这可能是Syn I C83片段导致认知功能障碍的重要原因之一。     

Thrombin activates the p21-activated kinase in pulmonary artery smooth muscle cells. Role in tissue factor expression

The p21-activated serine/threonine kinases (PAK) play an important role in a variety of cellular functions. However, their role in the smooth muscle response to thrombin, which is activated upon vascular injury and promotes vascular remodelling processes, is not resolved. Here we investigated the role of PAK in thrombin signalling and regulation of tissue factor (TF), the activator of the extrinsic coagulation cascade, in pulmonary artery smooth muscle cells (PASMC), the main cell type responsible for vascular remodelling in pulmonary hypertension. PAK was rapidly phosphorylated in response to thrombin. Thrombin and active PAKT423E phosphorylated p38 MAP kinase (p38MAPK), ERK1/2, phosphatidylinositol-dependent kinase-1 (PDK1) and protein kinase B/Akt (PKB) whereas kinase-deficient PAK1 prevented activation of these kinases by thrombin. In addition, kinase- deficient MKK3 inhibited activation of PDK1 and PKB by thrombin. Further, thrombin and active PAK1 induced TF expression and promoter activity while kinase-deficient PAK1 diminished thrombin-induced TF upregulation. Moreover, kinase-deficient MKK3, PDK1 and PKB inhibited thrombin- and PAK-dependent TF expression and promoter activity. Together these findings show that PAK is a critical element of thrombin signalling in PASMC which is involved in the regulation of TF expression by sequentially activating MKK3/p38MAPK, PDK1 and PKB. Thus, PAK may play an important role in promoting vascular remodelling processes in pulmonary hypertension.