Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin.

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and hemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalized with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases.

A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate.

BACKGROUND: Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. METHODS AND RESULTS: Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX_Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner. CONCLUSION: This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society.

Mechanistic insights into thrombin's switch between "slow" and "fast" forms.

Thrombin is a multifunctional enzyme that plays an important role in blood coagulation, cell growth, and metastasis. Depending upon the binding of sodium ions, thrombin presents significantly different enzymatic activities. In the environment with sodium ions, thrombin is highly active in cleaving the coagulated substrates and this is referred to as the "fast" form; in the environment without sodium ions, thrombin turns catalytically less active and is in the "slow" form. Although many experimental studies over the last two decades have attempted to reveal the structural and kinetic differences between these two forms, it remains vague and disputed how the functional switch between the "fast" and "slow" forms is mediated by Na+ cations. In this work, we employ microsecond-scale all-atom molecular dynamics simulations to investigate the differences in the structural ensembles in sodium-bound/unbound and potassium-bound/unbound thrombin. Our calculations indicate that the regulatory regions, including the 60s, γ loops, and exosite I and II, are primarily affected by both the bound and unbound cations. Conformational free energy surfaces, estimated from principal component analysis, further reveal the existence of multiple conformational states. The binding of a cation introduces changes in the distribution of these states. Through comparisons with potassium-binding, the binding of sodium ions appears to shift the population toward conformational states that might be catalytically favorable. Our study of thrombin in the presence of sodium/potassium ions suggests Na+-mediated generalized allostery is the mechanism of thrombin's functional switch between the "fast" and "slow" forms.

Thrombin is a selective inducer of heparanase release from platelets and granulocytes via protease-activated receptor-1.

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding.

OBJECTIVE: Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. STUDY DESIGN: Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. RESULTS: Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects. CONCLUSION: These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users. IMPLICATIONS: Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding.

Why Cannot Suction Drains Prevent Postoperative Spinal Epidural Hematoma?

BACKGROUND: Postoperative spinal epidural hematoma (POSEH) is different from spontaneous or post-spinal procedure hematoma because of the application of suction drains. However, it appeared that suction drains were not effective for prevention of POSEH in previous studies. The purpose of this study was to test our hypothesis that POSEH can be caused by hypercoagulability. METHODS: This was an experimental study. One hundred fifty milliliters of blood was donated from each of the 12 consecutive patients who underwent spine surgery and infused into 3 saline bags of 50 mL each. One of the 3 bags in each set contained 5,000 units of thrombin. All of them were connected to 120 ± 30 mmHg vacuum suctions: drainage was started 8 minutes after connection to the vacuum system for 12 normal blood bags (BV8) and 12 thrombin-containing blood bags (TBV8) and 15 minutes after connection for the remaining 12 normal blood bags (BV15). The amount of initial and remaining hematoma at 20 minutes, 120 minutes, and 24 hours after vacuum application were measured by their weight (g). The primary endpoint was the difference between BV8 and TBV8. The secondary end point was the difference between BV8 and BV15. RESULTS: The remaining hematoma in TBV8 was significantly greater than that in BV8 at all measurement points: 46.3 ± 12.4 vs. 17.0 ± 1.3 (p = 0.000) at 20 minutes; 33.0 ± 8.2 vs. 16.3 ± 1.2 (p = 0.000) at 120 minutes; and 26.1 ± 4.0 vs. 15.8 ± 1.6 (p = 0.000) at 24 hours after vacuum application. The remaining hematoma of BV15 was significantly greater than that of BV8 at all measurement points: 30.0 ± 12.0 vs. 17.0 ± 1.3 (p = 0.002) at 20 minutes; 24.2 ± 7.6 vs. 16.3 ± 1.2 at 120 minutes (p = 0.002); and 22.2 ± 6.6 vs. 15.8 ± 1.6 (p = 0.004) at 24 hours after vacuum application. CONCLUSIONS: With a suction drain in place, the amount of remaining hematoma could be affected by coagulability. Thrombin-containing local hemostatics and the length of time elapsed before the commencement of suction resulted in hypercoagulability, indicating these two factors could be causes of POSEH.

Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells.

Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present.

Simulated thrombin responses in venous valves.

OBJECTIVE: Venous thromboembolism frequently results in thrombi formation near or within the pocket of a venous valve due to recirculating hemodynamics, which has been largely attributed to hypoxia-induced tissue factor (TF) expression. Numerical models are now capable of assessing the spatiotemporal behavior of the TF-initiated coagulation cascade under nonuniform hemodynamics. The aim of this study was to use such a numerical simulation to analyze the degree and location of thrombin formation with respect to TF position in the presence of disturbed flow induced by an open venous valve. METHODS: Thrombin formation was simulated using a computational model that captures the hemodynamics, kinetics, and chemical transport of 22 biochemical species. Disturbed flow is described by the presence of a valve in the equilibrium phase of the valve cycle with leaflets in a fully open position. Three different positions of TF downstream of the valve opening were investigated. RESULTS: The critical amount of TF required to initiate a thrombotic response is reduced by up to 80% when it is positioned underneath the recirculating regions near the valve opening. In addition, because of the increased surface area of the open valve cusp in conjunction with recirculating hemodynamics, it was observed that thrombin is generated inside the valve pocket even when the exposed region of TF is downstream of the valve. CONCLUSIONS: The presence of prothrombotic surface reactions in conjunction with recirculating hemodynamics provides an additional mechanism for thrombus formation in venous valves that does not require direct damage or dysfunction to the valve itself.

Thrombin Activates Latent TGFß1 via Integrin αvß1 in Gingival Fibroblasts.

Transforming growth factor ß (TGFß) regulates cell proliferation, differentiation, migration, apoptosis, and extracellular matrix production. It also plays a pivotal role in the pathogenesis of gingival overgrowth. Thrombin is a key player in tissue repair, remodeling, and fibrosis after an injury, and it exerts profibrotic effects by activating protease-activated receptors. Connective tissue growth factor (CTGF or CCN2) modulates cell adhesion, migration, proliferation, matrix production, and wound healing. It is overexpressed in many fibrotic disorders, including gingival overgrowth, and it is positively associated with the degree of fibrosis in gingival overgrowth. In human gingival fibroblasts, we previously found that TGFß1 induced CCN2 protein synthesis through c-jun N-terminal kinase and Smad3 activation. Thrombin stimulates CCN2 synthesis through protease-activated receptor 1 and c-jun N-terminal kinase signaling. Curcumin inhibited TGFß1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGFß1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGFß-neutralizing antibody, TGFß type I receptor inhibitor SB431542, and Smad3 inhibitor SIS3 inhibited approximately 86%, 94%, and 100% of thrombin-induced CCN2 synthesis, respectively. Furthermore, blocking integrin subunits αv and ß1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGFß1 levels; however, similar effects were not observed for integrins αvß3 and αvß5. These results suggest that protease-activated receptor 1-induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin αvß1-induced latent TGFß1 activation and subsequent TGFß1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGFß1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGFß1 activation.

Modification of platelet proteins by 4-hydroxynonenal: Potential Mechanisms for inhibition of aggregation and metabolism.

Platelet aggregation is an essential response to tissue injury and is associated with activation of pro-oxidant enzymes, such as cyclooxygenase, and is also a highly energetic process. The two central energetic pathways in the cell, glycolysis and mitochondrial oxidative phosphorylation, are susceptible to damage by reactive lipid species. Interestingly, how platelet metabolism is affected by the oxidative stress associated with aggregation is largely unexplored. To address this issue, we examined the response of human platelets to 4-hydroxynonenal (4-HNE), a reactive lipid species which is generated during thrombus formation and during oxidative stress. Elevated plasma 4-HNE has been associated with renal failure, septic shock and cardiopulmonary bypass surgery. In this study, we found that 4-HNE decreased thrombin stimulated platelet aggregation by approximately 60%. The metabolomics analysis demonstrated that underlying our previous observation of a stimulation of platelet energetics by thrombin glycolysis and TCA (Tricarboxylic acid) metabolites were increased. Next, we assessed the effect of both 4-HNE and alkyne HNE (A-HNE) on bioenergetics and targeted metabolomics, and found a stimulatory effect on glycolysis, associated with inhibition of bioenergetic parameters. In the presence of HNE and thrombin glycolysis was further stimulated but the levels of the TCA metabolites were markedly suppressed. Identification of proteins modified by A-HNE followed by click chemistry and mass spectrometry revealed essential targets in platelet activation including proteins involved in metabolism, adhesion, cytoskeletal reorganization, aggregation, vesicular transport, protein folding, antioxidant proteins, and small GTPases. In summary, the biological effects of 4-HNE can be more effectively explained in platelets by the integrated effects of the modification of an electrophile responsive proteome rather than the isolated effects of candidate proteins.

Generalidades del sistema de la coagulación y pruebas para su estudio / Overwiew of the Coagulation System and laboratory tests for its study

En la década de los años sesenta, se describió la cascada de la coagulación como una secuencia de eventos enzimáticos iniciada por dos vías, la intrínseca y la extrínseca, las cuales convergían en una vía común para generar una enzima multifuncional, denominada trombina. La principal función de esta enzima consistía en transformar el fibrinógeno, en fibrina, una proteína que se polimeriza espontáneamente para formar la base estructural del coágulo. Posteriormente, se propuso el Modelo Celular según el cual la coagulación no es la consecuencia de vías de activación enzimáticas secuenciales, sino de una red de interacciones entre proteínas plasmáticas y transmembranas, así como, varios tipos celulares, que permiten la formación de complejos enzimáticos altamente eficientes con la finalidad de generar trombina. Esta revisión explica en detalle ambos enfoques, además, aborda las diferentes funciones que cumple la trombina dentro de la hemostasia y los mecanismos de inhibición que regulan la coagulación. Finalmente, se describen diferentes pruebas empleadas en la actualidad para evaluar la funcionalidad del sistema de coagulación, como: el tiempo de tromboplastina parcial activado, el tiempo de protrombina, el tiempo de trombina, el tiempo de reptilasa, el tiempo de coagulación por ecarina y el uso de sustratos cromogénicos para evaluar cada factor de la coagulación. Finalmente, dado a que la generación de trombina es clave dentro de la coagulación y a que el potencial de generar trombina puede indicar propensión a desarrollar eventos trombóticos o hemorrágicos, en este trabajo se presentan los métodos existentes para determinar la generación de trombina.

In the sixties, the clotting cascade was proposed, which describes the coagulation process as a sequence of enzymatic events initiated by two different pathways, the intrinsic and the extrinsic pathways, converging on a common pathway, to generate a multifunctional enzyme, thrombin, whose main function is to convert fibrinogen into fibrin, a protein that polymerizes spontaneously to form the building block of a hemostatic clot. Later, it was proposed a cell-based model of the hemostasis according to that coagulation does not occur as a consequence of linear sequential enzyme activation pathways, but rather via a network of simultaneous interactions between plasmatic and transmembrane proteins, as well as several cellular types, that allow the formation of highly efficient enzymatic complexes that lead to thrombin generation. In this review, we summarize these two approaches highlighting the functions of thrombin within the hemostasis and the inhibition mechanisms that regulate the blood coagulation. Moreover, we described different tests that are used to assess the function of the coagulation system, such as: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, ecarin clotting time, and the use of chromogenic substrates to evaluate individual coagulation factors. Finally, because of thrombin generation is a fundamental part of the blood coagulation and, an estimation of how well a particular individual can generate thrombin may correlate with either a risk of bleeding or thrombosis, we also include the existing methods to evaluate the potential of thrombin generation in an individual.

Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC).

Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (Lewis(X)), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system.

Colon Cancer Growth and Dissemination Relies upon Thrombin, Stromal PAR-1, and Fibrinogen.

Thrombin-mediated proteolysis is a major determinant of metastasis, but is not universally important for primary tumor growth. Here, we report that colorectal adenocarcinoma represents one important exception whereby thrombin-mediated functions support both primary tumor growth and metastasis. In contrast with studies of multiple nongastrointestinal cancers, we found that the growth of primary tumors formed by murine and human colon cancer cells was reduced in mice by genetic or pharmacologic reduction of circulating prothrombin. Reduced prothrombin expression was associated with lower mitotic indices and invasion of surrounding tissue. Mechanistic investigations revealed that thrombin-driven colonic adenocarcinoma growth relied upon at least two targets of thrombin-mediated proteolysis, protease-activated receptor-1 (PAR-1) expressed by stromal cells and the extracellular matrix protein, fibrinogen. Colonic adenocarcinoma growth was reduced in PAR-1-deficient mice, implicating stromal cell-associated PAR-1 as one thrombin target important for tumor outgrowth. Furthermore, tumor growth was dramatically impeded in fibrinogen-deficient mice, offering the first direct evidence of a critical functional role for fibrinogen in malignant tumor growth. Tumors harvested from fibrinogen-deficient mice displayed a relative reduction in cell proliferative indices, as well as increased tumor necrosis and decreased tumor vascular density. Collectively, our findings established a functional role for thrombin and its targets PAR-1 and fibrinogen in the pathogenesis of colonic adenocarcinoma, supporting tumor growth as well as local invasion and metastasis.

Mechanisms of early trauma-induced coagulopathy: The clot thickens or not?

Traumatic-induced coagulopathy (TIC) is a hemostatic disorder that is associated with significant bleeding, transfusion requirements, morbidity and mortality. A disorder similar or analogous to TIC was reported around 70 years ago in patients with shock, hemorrhage, burns, cardiac arrest or undergoing major surgery, and the condition was referred to as a "severe bleeding tendency," "defibrination syndrome," "consumptive disorder," and later by surgeons treating US Vietnam combat casualties as a "diffuse oozing coagulopathy." In 1982, Moore's group termed it the "bloody vicious cycle," others "the lethal triad," and in 2003 Brohi and colleagues introduced "acute traumatic coagulopathy" (ATC). Since that time, early TIC has been cloaked in many names and acronyms, including a "fibrinolytic form of disseminated intravascular coagulopathy (DIC)." A global consensus on naming is urgently required to avoid confusion. In our view, TIC is a dynamic entity that evolves over time and no single hypothesis adequately explains the different manifestations of the coagulopathy. However, early TIC is not DIC because an increased thrombin-generating potential in vitro does not imply a clinically relevant thrombotic state in vivo as early TIC is characterized by excessive bleeding, not thrombosis. DIC with its diffuse anatomopathologic fibrin deposition appears to be a latter phase progression of TIC associated with unchecked inflammation and multiple organ dysfunction.

Role of the extracellular signal-regulated kinase 1/2 signaling pathway in the process of thrombin-promoting airway remodeling in ovalbumin-allergic rats.

CONTEXT: Although it is recognized that thrombin plays a key role in airway remodeling during chronic asthma. In a previous study, we have proved that thrombin promotes airway remodeling via PAR-1 in OVA-allergic rats, but little is known about intracellular signaling pathway involved in the event. OBJECTIVE: In this study, we intend to explore the impact of pERK1/2 signaling pathway on the process of thrombin-induced airway remodeling in OVA-allergic rats. MATERIALS AND METHODS: A rat model of chronic asthma was set up by systemic sensitization and repeated challenge to OVA. The doses of thrombin, recombinant hirudin, PAR-1 inhibitor ER-112780-06, and pERK1/2 inhibitor PD98059 varied for different groups. The expression of pERK1/2 was analyzed by western blot and RT-PCR. Secretion of TGF-ß1 and IL-6 was detected by ELISA. RESULTS: The expression of pERK1/2 was higher in the airway of asthmatic rats than those of normal rats, and was significantly increased by thrombin treatment but decreased by thrombin-inhibitor treatment. Airway remodeling was enhanced by thrombin but weakened by pERK1/2 inhibitor. Expression of growth factors and IL-6 in asthmatic rats was significantly increased by thrombin treatment and decreased by thrombin-inhibitor treatment and pERK1/2 inhibitor treatment. CONCLUSION: These results suggest that ERK1/2 signaling pathway may play an important role in the process of thrombin-promoting airway remodeling in OVA-allergic rats, and pERK1/2 inhibitor effectively inhibits the process.

Thrombin down-regulates tissue factor pathway inhibitor expression in a PI3K/nuclear factor-κB-dependent manner in human pleural mesothelial cells.

Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the extrinsic coagulation cascade, and its expression is reported to be relatively stable. Various pathophysiologic agents have been shown to influence TFPI activity by regulating its expression or by modifying the protein. It is not clear how TFPI activity is regulated in normal physiology or in injury. Because thrombin and TFPI are locally elaborated in pleural injury, we sought to determine if thrombin could regulate TFPI in human pleural mesothelial cells (HPMCs). Thrombin significantly decreased TFPI mRNA and protein levels by > 70%. Thrombin-mediated down-regulation of TFPI promoted factor X activation by HPMCs. The ability of thrombin to significantly decrease TFPI mRNA and protein levels was maintained at nanomolar concentrations. Protease-activated receptor (PAR)-1, a mediator of thrombin signaling, is detectable in the mesothelium in human and murine pleural injury. PAR-1 silencing blocked thrombin-mediated decrements of TFPI in HPMCs. Thrombin activates PI3K/Akt and nuclear factor κB (NF-κB) signaling in HPMCs. Inhibition of PI3K (by PX-866) and NF-κB (by SN50) prevented thrombin-mediated TFPI mRNA and protein down-regulation. These are the first studies to demonstrate that thrombin decreases TFPI expression in HPMCs. Our findings demonstrate a novel mechanism by which thrombin regulates TFPI expression in HPMCs and promotes an unrestricted procoagulant response, and suggest that interactions between PI3K and NF-κB signaling pathways are linked in HPMCs and control TFPI expression. These findings raise the possibility that targeting this pathway could limit the ability of the mesothelium to support extravascular fibrin deposition and organization associated with pleural injury.

Disabled-2 is required for efficient hemostasis and platelet activation by thrombin in mice.

OBJECTIVE: The essential role of platelet activation in hemostasis and thrombotic diseases focuses attention on unveiling the underlying intracellular signals of platelet activation. Disabled-2 (Dab2) has been implicated in platelet aggregation and in the control of clotting responses. However, there is not yet any in vivo study to provide direct evidence for the role of Dab2 in hemostasis and platelet activation. APPROACH AND RESULTS: Megakaryocyte lineage-restricted Dab2 knockout (Dab2(-/-)) mice were generated to delineate in vivo functions of Dab2 in platelets. Dab2(-/-) mice appeared normal in size with prolonged bleeding time and impaired thrombus formation. Although normal in platelet production and granule biogenesis, Dab2(-/-) platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen in response to low concentrations of thrombin, but not other soluble agonists. Investigation of the role of Dab2 in thrombin signaling revealed that Dab2 has no effect on the expression of thrombin receptors and the outside-in signaling. Dab2(-/-) platelets stimulated by low concentrations of thrombin were normal in Gαq-mediated calcium mobilization and protein kinase C activation, but were defective in Gα12/13-mediated RhoA-ROCKII activation. The attenuated Gα12/13 signaling led to impaired ADP release, Akt-mammalian target of rapamycin and integrin αIIbß3 activation, fibrinogen binding, and clot retraction. The defective responses of Dab2(-/-) platelets to low concentrations of thrombin stimulation may contribute to the impaired hemostasis and thrombosis of Dab2(-/-) mice. CONCLUSIONS: This study sheds new insight in platelet biology and represents the first report demonstrating that Dab2 is a key regulator of hemostasis and thrombosis by functional interplay with Gα12/13-mediated thrombin signaling.

Altered plasma fibrin clot properties and fibrinolysis in patients with multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is associated with increased risk of venous and arterial thromboembolism. Formation of denser and poorly lysable fibrin clots is observed in patients with arterial and venous thromboembolism. We investigated fibrin clot properties and their determinants in MM patients. MATERIALS AND METHODS: Ex vivo plasma fibrin clot permeability, turbidity and susceptibility to lysis were evaluated in 106 MM patients at the time of diagnosis vs. 100 age- and sex-matched controls. MM patients had lower clot permeability (Ks ), compaction, indicating denser fibrin clots, impaired fibrin polymerization with longer lag phase and lower final turbidity (D-Dmax ), combined with hypofibrinolysis reflected by longer lysis time and slower rate of D-dimer release from fibrin clots (D-Drate ) compared with controls (all P < 0·001). RESULTS: Patients with IgG MM had lower Ks compared with IgA MM [5·9 (5·1-6·4) vs. 6·3 (5·9-7·2) 10(-9)  cm(2) ; P = 0·007] and longer lysis time compared with light-chain-disease patients [11·4 (10·9-12·3) vs. 10·7 (9·8-11·9) min; P = 0·022]. Of the fibrin variables, only Ks was significantly lower in patients with International Staging System (ISS) grade III than in those with ISS grade I and II [5·9 (4·9-6·6) vs. 6·2 (5·7-6·8) 10(-9)  cm(2) ; P = 0·015]. Multivariate analysis adjusted for age and fibrinogen showed that in MM patients elevated peak thrombin levels determine Ks and D-Dmax , while thrombin-activatable fibrinolysis inhibitor (TAFI) activity predicts Ks , t50% , D-Drate and lag phase. CONCLUSIONS: Our study demonstrates prothrombotic fibrin clot phenotype in patients with MM, with a significant impact of increased thrombin formation and TAFI activity.

New antithrombotics for secondary prevention of acute coronary syndrome.

Patients with acute coronary syndrome (ACS) usually receive acetylsalicylic acid plus an adenosine diphosphate (ADP) receptor inhibitor to reduce the long-term risk of recurrent events. However, patients receiving standard antiplatelet prophylaxis still face a substantial risk of recurrent events. Strategies involving 3 antithrombotic agents with different modes of action have now been tested. In Thrombin Receptor Antagonists for Clinical Event Reduction (TRA-CER), compared with standard care alone, bleeding complications including intracranial hemorrhage (ICH) were increased with the addition of vorapaxar, without efficacy benefit. In Trial to Assess the Effects of SCH 530348 in Preventing Heart Attack and Stroke in Patients With Atherosclerosis (TRA 2°P-TIMI 50), the addition of vorapaxar reduced recurrent events compared with standard care in stable patients with prior myocardial infarction. This study was terminated early in patients with prior stroke owing to excess ICH, though an increased risk of ICH or fatal bleeding was not detected in patients with prior myocardial infarction. The Apixaban for Prevention of Acute Ischemic and Safety Events 2 (APPRAISE-2) trial of standard-dose apixaban added to standard care in patients with ACS was also stopped early owing to excess serious bleeding. However, in Rivaroxaban in Combination With Aspirin Alone or With Aspirin and a Thienopyridine in Patients With Acute Coronary Syndromes (ATLAS ACS 2 TIMI 51), fatal bleeding or fatal ICH did not increase with low-dose rivaroxaban added to low-dose acetylsalicylic acid-based standard care compared with standard care alone. In that trial, a significant reduction of recurrent vascular events was shown with 3 antithrombotic regimens compared with standard care. Therefore, depending on drug dose and patient population, further reductions in recurrent vascular events after ACS may be possible in future clinical practice, with a favorable benefit-risk profile.