Formation of mutagens during the frying of Hawaiian fish: correlation with creatine and creatinine content

Compounds mutagenic toward Salmonella typhimurium strain TA98 in the presence of rat-liver homogenates (S9) were formed when fish flesh was fried at 199 degrees C. Three species of Hawaiian fish commonly consumed in Hawaii (skipjack tuna, Katsuwonus pelamis; yellowfin tuna, Neothunnus macropterus; and milkfish, Chanos chanos) were cooked in an electric skillet, along with samples of sole (Microstomus pacificus). Organic extracts of the fish were tested in the Ames Salmonella mutagenic assay using tester strain TA98 and S9. Basic organic extracts of fried, but not raw, samples exhibited significant mutagenicity. The levels of mutagenicity were also higher among the red flesh Hawaiian fish ('ahi, aku and awa) than with the white flesh sole. Creatine and creatinine contents were highest in the Hawaiian fish and lower in the sole. Creatine levels in the fish were 50-100 times greater than the creatinine content and varied from a high of 645 mg/100 g wet weight of fish for yellowfin tuna to a low value of 251 mg/100 g for sole. Mutagen levels are only approximately related to creatine/creatinine levels suggesting that other components contained in these fish may be as important as the guanidines in determining the levels of mutagen in the cooked fish.     

Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3

Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a translocation of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low - but not high - doses of ADP or thrombin which depends on α(IIb)β(3) integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.     

Isolation of a type IV collagen binding protein from human platelets.

In order to study the molecular basis of platelet interaction with collagen IV of the basement membrane separating the arterial endothelium from the underlying subendothelial connective tissue, the possibility of presence of platelet membrane protein with affinity to type IV collagen was examined by subjecting the platelet membrane extract to affinity chromatography on collagen IV-sepharose. Urea (4 M) eluate was found to contain a protein with an apparent mol. wt of 68 kDa. The radioiodinated protein was isolated and used to test its specificity. By dot blot assay on nitrocellulose disks and solid-phase assays, the 68 kDa protein was found to bind with high affinity to collagen IV. Lack of significant binding to fibronectin and laminin when compared to albumin control indicated its high specificity for collagen. The radioiodinated protein was inserted into egg yolk lecithin liposomes. While these liposomes attached to microtitre plates coated with collagen IV, there was no significant binding to fibronectin or laminin coated wells, suggesting the membrane associated character of the protein as well as its specificity for collagen. These results indicate that presence of a 68 kDa protein in platelet membrane which interacts with very high specificity to collagen IV.     

Fibrinolytic properties of activated FXII.

Activated factor XII (FXIIa), the initiator of the contact activation system, has been shown to activate plasminogen in a purified system. However, the quantitative role of FXIIa as a plasminogen activator in contact activation-dependent fibrinolysis in plasma is still unclear. In this study, the plasminogen activator (PA) activity of FXIIa was examined both in a purified system and in a dextran sulfate euglobulin fraction of plasma by measuring fibrinolysis in a fibrin microtiter plate assay. FXIIa was found to have low PA activity in a purified system. Dextran sulfate potentiated the PA activity of FXIIa about sixfold, but had no effect on the PA activity of smaller fragments of FXIIa, missing the binding domain for negatively charged surfaces. The addition of small amounts of factor XII (FXII) to FXII-deficient plasma induced a large increase in contact activation-dependent PA activity, as measured in a dextran sulfate euglobulin fraction, which may be ascribed to FXII-dependent activation of plasminogen activators like prekallikrein. When more FXII was added, PA activity continued to increase but to a lesser extent. In normal plasma, the addition of FXII also resulted in an increase of contact activation-dependent PA activity. These findings suggested a significant contribution of FXIIa as a direct plasminogen activator. Indeed, at least 20% of contact activation-dependent PA activity could be extracted from a dextran sulfate euglobulin fraction prepared from normal plasma by immunodepletion of FXIIa and therefore be ascribed to direct PA activity of FXIIa. PA activity of endogenous FXIIa immunoadsorped from plasma could only be detected in the presence of dextran sulfate. From these results it is concluded that FXIIa can contribute significantly to fibrinolysis as a plasminogen activator in the presence of a potentiating surface.     

A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein.

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

Compensatory growth in Alaska yellowfin sole, Pleuronectes asper, following food deprivation

During the early spring four groups of sub-adult Pleuronectes asper were fasted for either 0, 2, 4 or 6 weeks at the beginning of a 12-week experiment, then fed to satiation to examine their ability to compensate with faster growth after food deprivation. All fish increased their stored energy reserves markedly and at the end of the experiment all four groups had similar body energy content (J g⁻¹), length gains and dry weight to wet weight ratios. The groups of yellowfin sole fed continuously or fasted for 2 weeks gained the most weight, 25 and 24% respectively. Fishes fasted either 4 or 6 weeks exhibited significantly lower weight gains of 16 and 15% respectively over the 12-week experiment. Because of this disparity in weight gain the total body energy content of the continuously fed fish and those fasted for only 2 weeks increased by approximately 60 vs 46% or 35% for sole fasted for 4 or 6 weeks.
The experiment showed P. asper had a limited capacity for compensatory growth. When food was scarce yellowfin sole allocated energy preferentially to growth in length instead of weight. These findings may account for some of the interannual differences in mean length and weight at age for yellowfin sole from the Bering Sea where variations in extent and duration of ice cover and the boreal bottom water delimit the growing season.     

Cloning,characterization, and functional studies of a 47-kDa platelet receptor for type III collagen

A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction. A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins. Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column. The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns. Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner. We have defined two active peptides from the cloned deduced amino acid sequence. Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion. These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C_8 reverse phase HPLC. The purified activity, named longieornin, is a protein of moleeular weight 16 000 on SDS-PAGE under both reduced and nonredueed conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC_(50) was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effeetors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longieonin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intraeellar Ca~(2 ) level of platelets in response to collagen were com     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands fromHaemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C₈ reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggegation of platelets in plasma and of washed platelets (IC₅₀ was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar ca²⁺ level of platelets in response to collagen were completely eliminated by longicomin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better undentand the mechanism of collagen stimulation of platelets. Project supported by the National Natural Science Foundation of China (Grant No. 39170591).     

Binding of activated Factor XII to endothelial cells affects its inactivation by the C1-esterase inhibitor.

It is well known that activated Factor XII (FXIIa) and kallikrein are rapidly inactivated in plasma as a result of reaction with endogenous inhibitors. The purpose of this may be to prevent uncontrolled deleterious spreading and activation of target zymogens. Both FXII and the complex plasma prekallikrein/high molecular mass kininogen become activated when they bind, in a Zn2+-dependent manner, to receptors on human umbilical vein endothelial cells (HUVEC). The C1-esterase inhibitor (C1-INH) is by far the most efficient inhibitor of FXIIa. In the present study it has been investigated whether binding of FXIIa to HUVEC might offer protection against inactivation by C1-INH. It appeared that the relative amidolytic activity of purified FXIIa bound to the surface of HUVEC decreased according to the concentration of C1-INH in medium; however, the decrease was smaller than that measured for inactivation of FXIIa in solution. The secondary rate constant for the inactivation was 3-10-fold lower for cell-bound than for soluble FXIIa. The inactivation was found to be caused by C1-INH binding to cell-bound FXIIa. Accordingly, the amidolytic activity of saturated amounts of cell-bound FXIIa was reduced in the presence of C1-INH and was theoretically nonexistent at physiological C1-INH concentrations. Amidolytic activity was, however, present on HUVEC incubated with plasma indicating that the endogenous C1-INH did not completely abolish the activity of FXIIa generated during the incubation period. This supports the hypothesis that binding to endothelial cells protects the activated FXII against inactivation by its major endogenous inhibitor. Hence, the function of FXII may be localized at cellular surfaces.     

The involvement of cytoskeleton in the regulation of transbilayer movement of phospholipids in human blood platelets

Activation of human platelets by different activators resulted in a different extent of degradation of the cytoskeletal proteins actin-binding protein and myosin, as well as of the non-cytoskeletal protein P235. The highest extent of proteolysis was observed with Ca-ionophore A23187 and decreased on going from A23187 greater than collagen plus thrombin greater than collagen greater than thrombin = ADP. The same order of potency has been found previously ((1983) Biochim. Biophys. Acta 736, 57-66) for the ability of platelet activators to induce exposure of aminophospholipids in the outer leaflet of the platelet plasma membrane, and to stimulate platelets to become procoagulant. Degradation of cytoskeletal proteins as a result of platelet stimulation by collagen plus thrombin was prevented in the presence of dibutyryl cAMP or EDTA but not in the presence of aspirin. This also runs in parallel with platelet procoagulant activity. Moreover, platelets from a patient with a partial deficiency in platelet procoagulant activity revealed a diminished extent of degradation of cytoskeletal proteins upon platelet stimulation with collagen plus thrombin. It is concluded that alterations in cytoskeletal organization upon platelet stimulation may lead to alterations in the orientation of (amino)phospholipids in the plasma membrane, and may therefore play a regulatory role in the expression of platelet procoagulant activity.     

The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg++- dependent adhesion of platelets to collagen

We have purified the platelet membrane glycoprotein Ia-IIa complex by detergent solubilization and sequential affinity chromatography on Concanavalin A-Sepharose and collagen-Sepharose. The complex, which is identical to the VLA-2 complex of lymphocytes and other cells and contains subunits of 160 and 130 kD on SDS-PAGE, was labeled with 125I and incorporated into phosphatidyl choline liposomes. The liposomes, like intact platelets, adhered to collagenous substrates in an Mg++-dependent manner with a K'a(Mg++) of 3.5 mM. Little adhesion of the liposomes to collagen occurred when Mg++ was replaced by Ca++ or EDTA. Calcium ions inhibited the Mg++-dependent adhesion with a K'i(Ca++) of 5.5 mM. Liposomes containing the Ia-IIa complex adhered to substrates composed of types I, II, III, and IV collagen, but did not effectively adhere to substrates composed of type V collagen or gelatin. Adhesion to collagen was specific. The liposomes did not adhere to fibronectin, vitronectin, laminin, thrombospondin, fibrinogen, or von Willebrand factor substrates. The monoclonal antibody P1H5, which specifically immunoprecipitated the Ia-IIa complex, also specifically inhibited the Mg++-dependent adhesion of both platelets and Ia-IIa-containing liposomes to collagen substrates. These findings provide additional evidence that the platelet membrane Ia-IIa complex is the mediator of Mg++-dependent platelet adhesion to collagen and suggest that the VLA-2 complex may also function as an Mg++-dependent collagen receptor in other cells.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tickHaemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands fromHaemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C₈ reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggegation of platelets in plasma and of washed platelets (IC₅₀ was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar ca²⁺ level of platelets in response to collagen were completely eliminated by longicomin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better undentand the mechanism of collagen stimulation of platelets.     

The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen

We have previously shown that platelets adhere to collagen substrates via a Mg2(+)-dependent mechanism mediated by the surface glycoprotein Ia-IIa (human leukocyte very late activation protein 2, alpha 2 beta 1 integrin) complex. The adhesion is specific for collagen and is supported by collagen types I, II, III, IV, and VI. Several other members of the integrin family of adhesive protein receptors recognize discrete linear amino acid sequences within their adhesive glycoprotein ligands. Experiments with both intact platelets and with liposomes containing the purified receptor complex indicated that the alpha 2 beta 1 receptor recognized denatured type I collagen in a Mg2(+)-dependent manner. To further localize the binding site, the alpha 1 and alpha 2 chains of type I collagen were purified by gel filtration and ion exchange chromatography and tested as adhesive substrates. Both the alpha 1(I) and alpha 2(I) chains effectively supported Mg2(+)-dependent platelet adhesion. The purified alpha 1(I) collagen chain was then subjected to cleavage with cyanogen bromide, and the resultant peptides were separated by chromatography on carboxymethylcellulose. Only the alpha 1(I)-CB3 fragment supported Mg2(+)-dependent platelet adhesion. The monoclonal antibody P1H5 which recognizes an epitope on the alpha 2 subunit of the integrin receptor and which inhibits the adhesion of both intact platelets and liposomes bearing the purified receptor to collagen also inhibited platelet adhesion to the alpha 1(I)-CB3 fragment. These results indicate that the alpha 2 beta 1 receptor recognizes a sequence of amino acids present in the alpha 1(I)-CB3 fragment of type I collagen. An identical or similar sequence likely mediates binding of the receptor to other collagen polypeptides.     

Isolation and characterization of a platelet surface collagen binding complex related to VLA-2

A heterodimeric, Mg++-dependent, collagen binding protein has been isolated from platelet membranes. Electrophoretic properties and monoclonal antibody reactivity indicate that the heavy chain of the complex is platelet membrane glycoprotein Ia and that the light chain is glycoprotein IIa. Furthermore, the receptor appears to be identical with the recently defined VLA-2 complex found on activated T-lymphocytes, platelets and other cells. When incorporated into liposomes, the purified complex mediates the Mg++-dependent adhesion of the liposomes to collagen substrates. These observations suggest that the VLA-2 complex mediates cellular adhesion to collagen in platelets and possibly in other cells.     

Receptor- and phorbol-ester-mediated redistribution of protein kinase C in human platelets. Evidence that aggregation promotes degradation of protein kinase C.

Translocation of Ca2+/phospholipid-dependent protein kinase (PKC) activity from cytosolic to membrane fractions was assessed in washed human platelet suspensions. Phorbol myristate acetate (PMA) induced a rapid loss of PKC activity from the cytosolic compartment in stirred platelets, which was not accompanied by measurable increases in membrane-associated activity, but was paralleled by a decrease in total cellular enzyme activity (cytosol plus membrane). When platelet aggregation was prevented by not stirring, (i) cytosolic activity was decreased by PMA, (ii) significant and maintained (1-15 min with PMA) increases in membrane-bound PKC were detected, and (iii) the decline in total enzyme activity was markedly slower. In stirred platelets, total and specific inhibition of PMA-induced aggregation by a fibrinogen-derived peptide (RGDS, i.e. Arg-Gly-Asp-Ser) promoted maximal increases in membrane-associated PKC in the presence of PMA and completely prevented the loss in cellular activity. Thrombin and collagen both induced a decrease in cytosolic PKC and a loss of total activity, but a significant rise in membrane activity was seen only with collagen; ADP had no detectable effect on enzyme distribution. These results demonstrate an agonist-induced redistribution of PKC and indicate that platelet aggregation may play an important role in the proteolysis, and hence persistence, of membrane-associated PKC. This observation has implications for the potency and duration of PKC-mediated responses induced by agonists and exogenous PKC activators.     

Platelet C1q receptor interactions with collagen- and C1q-coated surfaces

We recently described specific binding sites for C1q on human blood platelets. Structural similarities between the amino-terminal of C1q and collagen have suggested that receptors for both molecules on platelets might be the same. The present study thus compared the interaction of purified C1q receptors (C1qR) and whole platelets with collagen- and C1q-coated polystyrene surfaces. Surfaces coated with BSA or gelatin served as controls. Purified 125I-labeled C1qR recognized both C1q- and collagen-coated surfaces in a divalent, cation-independent manner. This adhesion was inhibited by polyclonal or monoclonal (II1/D1) anti-C1qR antibodies. Although C1qR adhered preferentially to C1q-coated surfaces, adhesion to bovine and human type I collagen, as well as to human type III and V collagen, was also noted. In parallel studies, 51Cr-labeled platelets bound equally well to collagen- or C1q-coated surfaces, albeit in a magnesium-dependent manner. Partial inhibition of platelet adhesion was observed in the presence of RGDS, despite the inability of RGDS to modify C1qR interaction with C1q or collagen. Moreover, anti C1qR antibodies selectively inhibited platelet adhesion to C1q-coated surfaces, whereas antibodies specific for the GPIa/IIa collagen receptor (6F1) preferentially inhibited platelet collagen interactions. These data support the presence of distinct platelet membrane C1qR, which may cross-react with collagen, and suggest that C1qR are necessary but not sufficient for platelet adhesion to C1q-coated surfaces. Additional divalent cation and/or RGD-sensitive binding sites may participate.     

Dabigatran ameliorates airway smooth muscle remodeling in asthma by modulating Yes‐associated protein

Accumulating evidence indicates that thrombin, the major effector of the coagulation cascade, plays an important role in the pathogenesis of asthma. Interestingly, dabigatran, a drug used in clinical anticoagulation, directly inhibits thrombin activity. The aim of this study was to investigate the effects and mechanisms of dabigatran on airway smooth muscle remodeling in vivo and in vitro. Here, we found that dabigatran attenuated inflammatory pathology, mucus production, and collagen deposition in the lungs of asthmatic mice. Additionally, dabigatran suppressed Yes‐associated protein (YAP) activation in airway smooth muscle of asthmatic mice. In human airway smooth muscle cells (HASMCs), dabigatran not only alleviated thrombin‐induced proliferation, migration and up‐regulation of collagen I, α‐SMA, CTGF and cyclin D1, but also inhibited thrombin‐induced YAP activation, while YAP activation mediated thrombin‐induced HASMCs remodeling. Mechanistically, thrombin promoted actin stress fibre polymerization through the PAR1/RhoA/ROCK/MLC2 axis to activate YAP and then interacted with SMAD2 in the nucleus to induce downstream target genes, ultimately aggravating HASMCs remodeling. Our study provides experimental evidence that dabigatran ameliorates airway smooth muscle remodeling in asthma by inhibiting YAP signalling, and dabigatran may have therapeutic potential for the treatment of asthma.     

A fibronectin-binding glycoprotein from human platelet membranes.

Fibronectin ('cold-insoluble globulin') has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.