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Employees in the town of Skaneateles' highway department work with loud equipment day in and day out, and one has to question how their hearing is affected after years of being in such a noisy environment.
Town councilwoman Ellen Kulik said there state Department of Labor is now requiring yearly hearing evaluations for those employees and to have the tests completed by an audiologist.
"Right now the Department of Labor is only requiring it of the highway departments," she said.
However, there has been a request by the Safety Committee for the town to require others who work with heavy and loud machinery, like the parks employees who use the large mowers to cut the grass, to undergo the same examinations.
The question remains if the health department will come in and completely the screenings or if the town will need to find a trained and licensed audiologist. Regardless of who does it, Kulik said the screenings must be done at least for the highway department employees.
"We need to set up a program. If we don't, we'll be in violation of the Department of Labor," she said. "They want hearing screenings yearly."
If the Department of Health came in and provided the screenings at no cost, or if there were an audiologist available who could do the same, Kulik said it would be a "beautiful thing." If the town cannot find someone to complete the testing for no money, it could turn into a costly mandate.
"BOCES may provide that service for us," said councilwoman Nancy Murray. BOCES has a program for audiology which could cost the town very little if anything should they go through that program for the hearing tests.
According to Kulik, because the Department of Labor has told the town it needs to provide these tests for its employees, there may be reimbursement from the state at some point.
The board approved for the highway department to have the annual hearing tests completed by an audiologist.
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What You Need to Know About Cerebral Palsy »
"Cerebral palsy (CP) is the most common motor disability in childhood, and these children and their families need support. Professionals who serve young children, from early childhood educators to pediatricians, play an important role in identifyi"...
Read the What You Need to Know About Cerebral Palsy article »
Patients receiving iodinated intravascular contrast agents should be instructed to:
- Inform your physician if you are pregnant. (See PRECAUTIONS - Pregnancy)
- Inform your physician if you are diabetic or if you have multiple myeloma, pheochromocytoma, homozygous sickle cell disease, or known thyroid disorder. (See WARNINGS.)
- Inform your physician if you are allergic to any drugs, or food, or if you have immune, autoimmune or immune deficiency disorders. Also inform your physician if you had any reactions to previous Injections of dyes used for x-ray procedures. (See PRECAUTIONS, General.)
- Inform your physician about all medications you are currently taking, including non-prescription drugs (over-the-counter) drugs, before you have this procedure.
Last reviewed on RxList: 2/26/2009
This monograph has been modified to include the generic and brand name in many instances.
Oxilan FDA Prescribing Information
Additional Oxilan Information
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Archive for Thursday, December 14, 2006
Agape Circle meeting
The Agape Circle of De Soto United Methodist Church will meet at 7 p.m. Dec. 21 at the home of Diane Lavenburg.
The group will discuss Chapter 10 from "The Christian's Secret of a Happy Life."
Guests are welcome to attend.
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HDMI V1.4 HDMI Male to Male Connection Cable - Blue (300cm)
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Hola buenos días. Lo utilizo para conectar mi portátil a la TV y funciona perfectamente. Lo recomiendo. Un saludo.
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Sands' companion to The Countess abounds with her hallmark humor and sensuality. Sands cleverly juxtaposes the stories in her duet from differing points of view, making this addition fresh and delightful.
Her sister's marriage had saved the family from ruin, but with their father's gambling debts mounting Suzette must find a husband and claim her inheritance. What she needs is a poor man in want of a wealthy wife, but with her sister's dead husband suddenly alive Suzette has a problem. When Daniel Woodrow rescues his best friend and they return to England, his goal is to help find a traitor, he never expects to encounter a bluntly outspoken young woman — she fascinates him. When Suzette makes an outrageous proposal that they marry Daniel readily accepts, once he gets rid of a dead body. If a corpse isn't enough of a problem, they find themselves racing cross country after a blackmailer, trapped in a carriage accident and captured by a villain before everything can be sorted out. (AVON, Mar., 380 pp., $7.99)
Reviewed by:
Kathe Robin


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Citi's Steven Englander On Black Swan Fatigue, And Why It Is Time To Hedge Tail Risk Again
From Citi's Steven Englander
Black Swan Fatigue and Under-Provisioning for Fat Tails
Tomorrow is the second anniversary of the S&P having begun its rebound from its cycle low (see the figure below)
We also see historical (and in some cases implied) volatility in many currency pairs at post-crisis lows (see chart below). We plot 1-month realized AUDJPY vol below because it is typically a metaphor for high volatility, but even this cross appears tame. A similar pattern is reflected in many G10 currency crosses.
Where do the Black Swans comes in? For much of this two year period of S&P recovery, the clever thinking among investors has been to fade the rebound. This is as much true of FX investors as in other classes. Generically, the arguments have been that the forces driving the recovery are fragile, that many fundamental issues in global financial markets are unresolved, and that policymakers have deferred rather than resolved problems.
It's hard to argue with this line of analysis except that it has been dead wrong in market terms. Our conjecture is that investors are tired of being told to hedge against risks that have not emerged, and where the positions end up as costly in terms of premium paid out. (And it is an obvious truth that the kids outside playing in the snow without sweaters and scarves seemed to have much more fun than those of us who were bundled up.) Indeed the downward trend in vol may reflect that too much optionality was bought to hedge risks that didn't happen and now it is being disgorged back into the market.
In recent weeks we have been arguing that tail risk remains and is unusually realistic given the potential sources of shocks. We have also found that in many cases clients are reluctant to buy into these fat tail scenarios (actually 'reject' may more accurate than ' are reluctant' ). The argument is basically that they have heard it many times before in the last two years and asset markets have continued to flourish.
We would argue that the correct approach is to ask whether the macro risks looking ahead are those that can be dealt with using the policy tools at hand. If not, the tail risk scenarios are more concrete. Conversely if the asset market bounce and rebound in high-beta currencies over the last two years can be explained largely as the result of massive liquidity injections globally and significant fiscal policy ease, the question looking ahead is whether these policy tools will be equally effective in dealing with the future shocks.
Consider the obvious potential risks: an oil shock, commodity prices and advancing headline inflation in the context of growing concerns about social and political disruption. We would make the case that the risk is high that if any of these shocks were to move to centre stage, it would be harder for policy makers to deal with them because printing and spending money is not quite the solution to these problems that it was to the financial shock of late 2008 and early 2009.
… which brings us back to Black Swan fatigue. When we see currency vols so cheap in a world that appears to be so risky and in which policymakers do not hold a terribly strong hand, it makes a strong case for hedging tail risk, even if FX price action is apparently indicating otherwise.
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swan tail bitches
Yes and once you've committed to tail risk management, you cannot and likely should not stop, until your investment thesis/activity has ended. This should be done no matter what the cost, as long as you don't go broke doing it, harr harr.
Good point, and it does cost, but put spreads are fer cheap if you do your homework. They also help you sleep at night;)
HR, very important, no doubt. Time is major factor. Big first mistake is to not buy enough (time). In other words, roll forward deep, barbell and participate in upside with whatever "carry asset" you prefer etc....Been sleeping well lately, thanks.
The Fed has a hammer and everything looks like a nail to them... the one trick pony will continue to print even if it solves nothing.
Its all rigged...
We have treasonous bankers running our system...
Oil, Silver, Gold all going to be rising and especially food prices. America will be like all other countries, and we will revolt... not by choice but by neccessity.
i.e. Hedge your long CHF long CAD and...even, gasp, long EUR positions.
It's taking longer for the banksters to get the rube's watching them play 3 card monty to ante up.
US ask Saudi's to arm Libya's Guerrillas......
and congrats to Zero Hedge on time mags list on best financial blogs..
""US ask Saudi's to arm Libya's Guerrillas......"'
Single best way to win a revolution?
...Starve the Government...
It is impossible to starve government if the FED exists to print money an buy debt. That's why the goverment will never willingly return to a gold standard. If people had to pay for government out of pocket, things would be different.
If people had to pay for government out of pocket, things would be different.
This is so true. While we rail against the Fed, and deservedly so, it is likewise the Federal Government that deserves our wrath and focus. The Fed and the government are mutual addicts and enablers, each feeding the other's habit. They are co-dependents and must simultaneously be brought under control.
They will fight to their destruction before giving up, the battle is not even begun.
I second the congrats to Zero Hedge for all their fine reporting. If not for them and a very, very few other sites we would all be dancing in the dark. Hat tip!
Well said, and not an easy pace. None of us has complained about a slow news day in a coon's age. No offence, Rocky. We all know you are a spring chicken at heart.
An environment in which hedging the black swan type future has become unpopular, is exactly the environment in which you want to be spending money on cheap Puts.
Compliments of Jesse...
"The weakness with this US Dollar DX index is that it is highly weighted to the developed economies of Europe and Japan. As such it may not reflect erosion of dollar purchasing power vis a vis the BRICs, and external measures such as gold, oil, and silver. It may be masked by the mutual weakness of central banks all inflating their currencies in unison."
"Rather than rallies through economic vitality and recovery, the dollar rallies have been marked by relative declines primarily in the euro on their sovereign debt problems. It is almost like a couple of drunks leaning on each other for support, except that the US is picking the Eurozone's pockets while they do it."
Remainder of comments here... http://jessescrossroadscafe.blogspot.com/
Here is a black like oil swan:
"Black Swan Fatigue" LOL. ZHers should seriously consider creating a 'ZH' terms dictionary.
Tonight's Movie:
Dr. StrangeBen, or: How I Learned to Stop Worrying and Love Inflation
Look out for my upcoming book, "Black Crow: The Mystification of the Thoroughly Mundane"
To wear the mermaid wedding dresses could be called the dream of every woman. I think whoever you are, to get married with the man your love is a holy thing. And we all firmly believe that marriage will not kill your love, on the contrary, wedding dress is just the beginning. So the bridesmaid Dresses chooses the wedding dresses carefully. Naturally, they want to become the most beautiful bridesmaid dresses and the unique sheath wedding dresses. The dresses in our online column wedding dresses store are their best choices.
May have its groove back.
Despite market intervention/QE - natural market forces can not be stopped – only delayed.
When the market does reassert control – the reaction (the overdue correction) may be even more extreme due to that delay.
And some may erroneously refer to this as a Black Swan since it's 'unexpected'.
However they will be wrong because major market events can be predicted by some analysts.
We need your help,come to us and make your voting:http://trendybull777.blog.com/2011/02/21/hello-world/
tell it to friend,we need to rich every interesting one to make his vote for FED existence


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Robert Bushnell Ryan (November 11, 1909 – July 11, 1973) was an American actor who often played hardened cops and ruthless villains.
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May 26, 2015
Dash cam shows moment plane crashes on Ga. interstate
|New police dashcam video shows the aftermath of a deadly plane crash on an Atlanta-area highway earlier this month.
April 28, 2015
A very lucky landing
|This Canadian paratrooper caught a lucky break and managed to land without injury after being dropped into trees from a C-130.
February 19, 2015
Fla. cops save autistic boy's birthday after no one shows up
|Police and firefighters in Florida reached out to a boy and his mother after a post on social media caught the attention of the local community.
February 09, 2015
Sniper kills Austin man who opened fire on police helicopter
|27-year-old Sawyer Flache opened fire on the helicopter as it arrived – resulting in a 30 minute standoff.
January 05, 2015
Chopper captures motorist's failed attempt at escaping police
|Police in England were able to keep an eye on a group of speeding motorists, leading to a successful arrest, thanks to aerial footage from a police helicopter.
January 02, 2015
Calif. harbor patrol officer killed in Catalina storm
|Tim Mitchell was fighting to protect residents and save boats during the storm when he attempted to jump to shore.
September 26, 2014
PA-HART - Vigilant Guard Exercise
|A Pennsylvania-HART rescue technician (RT) is lowered from a UH-60 Blackhawk to an injured victim. Once on scene, the technician completes an evaluation and determines a course of action. He places a rescue strop around the victim and communicates with the Blackhawk via radio for the hoist hook to be lowered. Once the technician and victim are attached to the hoist hook, the RT signals and they are hoisted to the Blackhawk.
June 09, 2014
Ariz. police helicopter crashes, pilots OK
|In the 15 seconds between complete engine failure and hitting the ground, officers were able to locate a field and make a hard landing.
March 04, 2014


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Janet Kaniki
Update on Janet KanikiJanet is a single woman. She has been operating a retail store where she provides M-pesa (mobile phone money transfer) services for the last four years as a Safaricom Agent.
Previously, she took a loan of KES 20,000 which she managed to pay back successfully. This is her third loan with SMEP DTM. She plan to use the loan of KES 40,000 as M-pesa float for the M-pesa services. Janet looks forward in the future to expanding her business.
Previous Loan DetailsJanet, 31 years old, is a single woman. She has no children yet. For the past one year, she has been selling mobile phones, phone accessories, and providing phone charging services. In addition, she is employed as a Safaricom agent. Sometimes in her business she faces a challenge of delayed ope... More from Janet Kaniki's previous loan »
Additional Information
Concurrent and Successive Loans
Our Field Partners often work with borrowers over a series of loans as the borrowers build credit, take out bigger loans, and expand their businesses. In order to make it easier for our Field Partners to post loans for borrowers who have been listed on Kiva before, we allow them to post successive and concurrent loans for their Kiva borrowers. This means that our Field Partners are able to post a borrower's second, third, etc., loan on Kiva without having to re-enter all of the borrower's information.
This borrower has been listed on Kiva before, so you'll see an updated loan description, as well as excerpts of the original descriptions from earlier loans. Most borrowers take out loans consecutively, meaning that they receive a second loan after having repaid the first. However, sometimes our Field Partners give out concurrent loans, allowing borrowers to take out one primary loan and a secondary "add-on" loan along with it. These "add-on" loans are typically smaller than the borrower's primary loan and serve a different purpose. Because Field Partners can now post loans as successive and concurrent loans, you will be able to track borrower progress over time and see the various ways a borrower is working with our Field Partners through funds from Kiva's lenders.
About Kenya
1071View loans »
Success!! The loan was 100% repaid
- Repayment Term
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- Repayment Schedule
- Monthly
- Pre-Disbursed:
- Feb 25, 2013
- Listed
- Mar 23, 2013
- Currency Exchange Loss:
- Possible
- Ended:
- Mar 17, 2014


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March 26, 2014
Bryan Holt
Make it five days of installation for Florida's renovated offense. The Gators remain under construction on the side of the ball they need to improve most, but that doesn't mean there aren't positives to point out as well. Here's a look at where the offense was at each position Wednesday. ...More... To continue reading this article you must be a member. Sign Up Now!
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Products page
Here you will find our Border Fine Arts catalogues, retirements and the details for all of the products featured on the website in one place.
Also this is now home to our Where to Buy lists and, well, anything else product related that we think you might like to know!
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To ensure that yours is painted to look like your pet they will need plenty of good photos of the horse, dog or cat, which clearly show any special markings. The cost of the process is 30% on top of the normal retail price and it takes approximately 4–6 weeks.
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Kaska Goose Lodge, Hudson Bay Lowlands, N Manitoba
From $1,125 / night
Description from the owner
Description from the owner
Bedrooms: 4/
Bathrooms: 2/
Sleeps: 8/
Type: Resort
This fully-modern guest cabin faces southeast, overlooking the Kaskattama River in northern Manitoba. The large common room has a cathedral ceiling and a full wall of floor to ceiling picture windows in the front wall. A wood-burning glass-doored energy efficient stove provides heat for the main cabin area.
There is a mud-room with a shotgun rack at the cabin entrance so hunters can safely store their guns after their day of hunting and take off their gear so they don't track dirt into the main cabin.
The cabin is split into two "wings" - each with two bedrooms and two bathrooms. One bathroom has a shower and the other a tub-shower. Each bedroom ...Read more
About the owner
About the owner
Randy Duvell
Randy Duvell
- Response rate : 85%
- Response time: A few hours
- Calendar updated: 14 months ago
Overall rating:
Additional Location Information
Kaskattama Safari Adventures is locate 650 miles NE of Winnipeg, Manitoba, where goose hunters' dreams are transformed into reality. Kaska's lodge and cabins are nestled along the crystal-clear Kaskattama River only one mile from the Hudson Bay coast near the northeastern tip of Manitoba. The 5,000 acre Kaskattama River delta provides an ideal habitat for thousands of Greater and Lesser Canada, Blue, Snow and Ross' geese to nest and stage before beginning their long journey south.
Originally built by the Hudson Bay Company as a fur-trading outpost in 1923, Kaska has metamorphosed into a comfortable retreat for discerning wing shooters.
Accommodating a maximum of only sixteen guests in two modern guest cabins, Kaska offers hunters not only a unique hunting experience, but a real adventure!
although Kaska Goose Lodge is totally remote in the wilds of the Hudson Bay Lowlands, it is readily accessible for our guests from virtually anywhere in North America. Delta, Air Canada / United Airlines as well as WestJet / American Airlines all have excellent daily service between the U.S. and Winnipeg - our gateway city. Transportation between Winnipeg and our private airstrip at Kaska Goose Lodge is either via a combination of scheduled and charter air carriers or private charter. We suggest our guests overnight in Winnipeg on their way in and out of the lodge.
Heli-hunting adds a complete new dimension to the Kaska experience. Our Bell Long Ranger helicopter quickly and safely transports hunters to nearby, but previously inaccessible staging areas where vast flocks of snow geese congregate. The helicopter also provides exceptional opportunities to see and photograph polar bears in their natural habitat.
Kaska is not just about goose hunting, as many species of ducks as well as rock and willow ptarmigan make the Kaskattama River delta their home. For guests wanting a diversion from hunting – within a short walk of the lodge the Kaskattama River boasts some trophy-quality sea-run brook trout that will test your mettle as a fisherman.
Kaska is also a naturalist's dream. The unique ecosystem features a striking and abrupt transition from spruce forest to tundra along the edge of the Hudson Bay. An outstanding habitat for polar bears, caribous, moose, black bear, Arctic fox, wolves, raptors, tundra swans and shorebirds, this frgile ecosystem offers unparalleled nature-viewing opportunities.
As the sun sinks below the horizon signalling the end of another day, the cheery lights of the main lodge and aromas foretelling a sumptuous dinner beckon you to join the camaraderie of your friends and other sportsmen in the comfort of the lodge.
Your day ends relaxing by the warmth of a crackling wood fire, sipping a fine cognac, recounting the day's adventures and admiring the awesome beauty of the northern lights dancing in the night sky. We invite you to experience the adventure of Kaska Goose Lodge – you owe it to yourself!
More About This Location
Beach or lakeside relaxation, Rural retreats, Waterfront


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Too crumbly
Posted by Reviewer2717476 on Sep 27, 2013
when I got the product most of the pills where not whole they were crumbly. still worked however I had to use several pices to get the same result. So unable to tell the dosage. Not a good thing
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Hassan Rouhani's government will not issue permits for women to sing solo, Ali Jannati, the minister of Culture and Guidance has declared.
[ . . . ]
Jannati remarks followed statements by two senior clerics in Qom announcing that women
cannot sing in Iran. Nouri Hamedani and Makarem Shirazi in their lectures in Qom protested an album that had songs of a female singer, state-run news agency website Tabnak reported on February 4.
Nouri Hamedani said in his lecture: There is no problem with women talking, but "women singing cannot become common and we will stop it.
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The Fox Hole
We open the door to crinkled, old, sodden carpet, and through the doorway we spot two mangy pool tables to the left, and to the right a shuffleboard table and a brass-plated bar the whole thing is this old, pounded brass plate, like gold-colored tin all over the bar. Awesome.
Dee the bartender is seasoned and just hired from the defunct Thunderbird Lounge up the road; she's from Ohio and brings some personality and warmth to the place.
We settle in and order a beer, which shows up quickly in a subzero frosty mug. We notice the centerpiece of the bar, a nice fish tank with a half a dozen or so of the little guys looking out at us. We ask Dee if she names them and she says, "Nah, but the big one on the bottom is the prettiest." The big guy next to us chimes in loudly, "Yeah, the shit-sucker!" Dee laughs and the big guy says, "Why is it that the prettiest one in there is the one that just sits around and eats shit all day... I don't get it."
Well, as we look around at these folks and the drunken grins and swaggers in this place, we get it loud and clear: This is the prettiest place we've found ourselves, in a long time.
But, perhaps, not for long. You'd better get over to The Fox Hole soon word is the place has new owners, and they already made the men's room "nice," and we guess they're looking to add new carpet soon... Giddy up before they ruin the charm up in the joint.
Location Details
More Bars & Clubs Awards
BEST PLACE FOR A SCOTCH AND A BASEBALL GAMEThe Club Lounge
BEST PIT STOP TO SEE BIKINI BABESCherry Lounge & Pit
BEST LOCAL BAND TO GET SOME NATIONAL RECOGNITIONPsychostick
BEST BAND WHO USE MOM'S POTS AND PANSTent/City


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Jane Wyman (born Sarah Jane Mayfield; January 5, 1917 – September 10, 2007) was an American singer, dancer, and film/television actress. She began her film career in 1932 and her work in television lasted into 1993. She was a prolific performer for two decades. She received an Academy Award for Best Actress for her performance in Johnny Belinda (1948), and was a three time winner of The Golden Globe, she achieved a new level of success as Angela Channing in the 1980s prime time soap opera Falcon Crest.
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'rHE JOURNAL Of BIOLOGICAL CHEMISTRY I C ! 1991 by The American Society for Biochemistry and Molecular Biology. Inc. VOl. 266, No. 19, Issue of July 5, pp. 12329-12336, 1991 Printed in U.S.A. Inhibition of Cell Surface Receptor-bound Plasmin by az-Antiplasmin and a2-Macroglobulin* (Received for publication, January 29, 1991) Scott W. Hall$, John E. Humphriesj, and Steven L. Goniasll From the Departments of Pathology, Biochemistry, and Internal Medicine, University of Virginia Health Sciences Center, Charlottesuifie, Virginia 22908 The purpose of this investigation was to characterize the reaction of az-antiplasmin (a2AP) and a2-macro- globulin (azM) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 p4 "C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plas- min) to C6 cells were 0.9 p~ and 2.6 X lo6 sites/cell. The dissociation rate constants (kOff) for '251-plasmin were 9.7 X and 4.0 X presence and absence of 0.3 p~ DIP-plasmin, respec- tively. Similar constants were determined for minogen and 1251-DIP-plasmin. Neither azAP nor aZM affected the dissociation of DIP-plasmin. C6 cell-asso- ciated '"I-plasmin reacted slowly with azAP; however, the inhibition rate constants exceeded the kOff. azAP- plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reac- tion of azAP with cell-associated enzyme pathway 2). High concentrations of azAP favored path- way 2. C6 cell-associated plasmin was also protected from inhibition by a2M. While the same pathways were probably involved in this reaction, azM was less tive than azAP as an inhibitor of nondissociated plas- min (pathway 2). When C6 cell-bound plasmin reacted with azAP, a2AP-plasmin complex was recovered pri- marily in the medium, suggesting dissociation of com- plexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were per- formed at 22 " and 37 "C, confirming the conclusions of the 4 "C studies. Comparable results were also ob- tained using HUVEC cultures. These studies demon- strate that cell-associated plasmin is protected from inhibition by a2M as well as azAP. At least two reaction pathways may be demonstrated for the inhibition plasmin that is initially receptor-bound; however, nei- ther pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface. ~) to C6 cells at s" at 4 "C in the '251-plas- (reaction effec- of * This work was supported by National Heart, Lung, and Blood Institute Grant HL45786 and by the Pew Scholars Program in the Biomedical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. j: Supported in part by Medical Scientist Training Program Grant GM 07267. I Supported in part Grant 1T32AM07509. ll Recipient of a Research Career Development Award (HL02272). To whom correspondence should he addressed University of Virginia Health Sciences Center, Departments of Pathology and Biochemistry, Box 214, Charlottesville, VA 22908. Tel.: 804-924-9192; Fax: 804-924- 8060. by National Institutes of Health Training The enzyme plasmin plays an important role in fibrinolysis, cell migration, tissue remodeling, angiogenesis, tumor inva- sion, and metastasis (1, 2). Most of these processes involve a cascade of serine and metalloproteinases (3-5). The activity of each proteinase may be localized and regulated by nonco- valent binding interactions with other macromolecules in the basement membrane and on cell surfaces. Specific cellular receptors for urokinase-type plasminogen activator (uPA),' tissue-type plasminogen activator, and plasminogen or plas- min have been identified in a wide variety of cell types (6-8). These receptors assemble the fibrinolytic system on the cell surface and enhance plasminogen activation. Plasminogen cell surface receptors seem to be ubiquitously expressed; however, the molecular nature of these binding sites remains incompletely understood. In the cell types ex- amined to date, certain receptor properties are (8). Plasminogen or plasmin binding is specific and saturable. Plasmin(ogen) endocytosis does not occur to a significant degree. Lysine or lysine analogues kringle domains of plasminogen prevent receptor binding. In addition, the receptor binding parameters are comparable in different cell types. For G1u'-plasminogen, the K,, is typically in the range of 1.0-2.0 p~,and the binding capacity high ( 104-107 sites/cell). The plasminogen-receptor interac- tion is conserved across species lines; plasminogens isolated from different mammalian species compete equivalently for receptors on rat C6 glioma cells and hepatocytes (9). Recent evidence suggests that the plasminogen receptor may represent a number of structurally divergent molecules with similar plasminogen binding activity (8). In some cell systems, plasminogen receptor binding increases the catalytic efficiency of plasminogen activation or promotes the conver- sion of single-chain uPA into the 10). A consistently observed property of plasmin bound to cell-surface receptors is resistance to inhibition plasmin (cuzAP) (11-13). In vitro and in the circulation, a2AP is the primary proteinase inhibitor regulation (14). The association rate constant for the reaction of plasmin with aaAP is 2 X 10' M" s" (15). In cell culture systems, receptor-bound plasmin reacts azAP; however, rate constants have not been determined (11- 13). Inactivation of receptor-bound plasmin by asAP may occur via two pathways: direct reaction ' The abbreviations used are: uPA, urokinase-type plasminogen activator; ru,AP, <u2-antiplasmin; a,M, n,-macroglobulin; PNPGB, p- nitropheny1p'-guanidinohenzoate hydrochloride; DIP-plasmin, diiso- propyl phosphoryl plasmin; Hepes, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid; HUVEC, human umbilical vein endothelial cells; EBSS, Earle's Balanced Salts Solution; acid; u-PAR, urokinase-plasminogen activator receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PAI, plas- minogen activator inhibitor; Pm, plasmin. well conserved that interact with the (Elmax) is active two-chain enzyme (8, by a2-anti- responsible for plasmin less readily with of the a2AP with EACA, c-amino-n-caproic 12329
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12330 Inhibition of Receptor-bound Plasmin receptor-bound plasmin at the cell surface, or plasmin disso- ciation followed by rapid inhibition in the solution phase. These pathways have not been characterized independently. a2-Macroglobulin (azM) is a second rapid inhibitor of plas- min. The rate constant for the reaction is approximately 5.0 x IO5 M-' s-'. When plasmin is bound to histidine-rich glycoprotein (16), or when elastase is bound to elastin (17), the proteinase inhibitory activity of a2M is only slightly decreased. Trypsin covalently coupled to the surface of sheep red blood cells is also inhibited by azM (18). There- fore, azM may present a significant inhibitory challenge to plasmin in a2AP-resistant environments. The reaction receptor-bound plasmin with azM has not been studied. The purpose of the present investigation processes involved in the regulation of cell-associated plas- min. Initially, we studied the rate of plasmin dissociation from cellular receptors. In subsequent studies, we characterized the reaction of receptor-bound plasmin with a2AP and a2M. All of these experiments were performed i n uitro using cultures of rat C6 glioma cells. Plasminogen receptors on share many common properties with the receptors expressed by endothelium, hepatocytes, and monocytes (binding affin- ity, effect of lysine, protection of plasmin from azAP) (13, 19). Therefore, the C6 cell was considered an appropriate model for plasmin inhibition experiments. The major results of this investigation were confirmed using cultures of human umbilical vein endothelial cells (HUVECs). In addition, ex- periments were designed to distinguish between two possible pathways for the inhibition of plasmin that is initially recep- tor-bound: 1) dissociation of cell-associated plasmin, followed by rapid inhibition in solution, and receptor-bound plasmin with proteinase inhibitors at the cell surface. of plasmin with a2M of was to characterize C6 cells 2) direct reaction of EXPERIMENTAL PROCEDURES Materials-p-Nitrophenyl (PNPGB) and diisopropylphosphorofluoridate were purchased from Sigma. o-Val-L-Leu-L-Lys-p-nitroanilide from Kabi Vitrum. NalZ5I was from Amersham Corp., and Iodobeads were from Pierce Chemical Co. Purified Proteins-Glu'-plasminogen zen human plasma as previously described (12). The preparations consisted of a mixture of carbohydrate variants, type I and 11, with variant I1 present in slight excess. As previously described, differences in the binding of plasminogen carbohydrate isozymes to C6 cells in culture are minimal (13). Purified against 10 mM Hepes, 50 mM sodium acetate, pH 7.4. Plasmin was generated by incubating plasminogen (3.0-5.0 pweight uPA (20 nM) (Calbiochem) for 15 min at 37 "C. Diisopropyl phosphoryl plasmin (DIP-plasmin) was prepared by incubating plas- min twice with 10 mM diisopropylphosphorofluoridate for 1 h at 22 "C. Excess diisopropylphosphorofluoridate was removed by di- alysis against 20 mM sodium phosphate, 154 mM NaC1, pH 7.4. The final preparation retained less than 0.1% of the original activity as determined by S-2251 hydrolysis rate. Plasmin was also inactivated by incubation with a 2-fold molar excess of aprotinin. a,AP was purified according to the procedure of Wiman (20). a2M was purified by the method of Imber and Pizzo (21). Radioiodination-Plasminogen was radioiodinated using either Io- dobeads (Pierce) as previously described (13) or a modified chlora- mine-T method (22). Specific activities ranged from 0.5 to 1.5 pCi/ pg. "511-Plasminogen was activated with uPA as described above. I2'I- Plasmin activity was decreased by less than 5% compared with the nonradiolaheled enzyme as determined by S-2251 hydrolysis rate. By SDS-PAGE under reducing conditions and autoradiography, plasminogen preparations demonstrated =90,000), indicating that there was no significant contamination with plasmin. After activation with uPA, greater than plasminogen was converted into the two-chain form characteristic plasmin. n2AP and a2M were radioiodinated using Iodobeads. The specific activities were 0.2 and 1.0 pCi/pg for a2AP and a2M, respectively. p'-guanidinobenzoate hydrochloride hydrochloride (S-2251) was was purified from fresh fro- Glul-plasminogen was dialyzed ~) with low molecular "'1- only a single band (Mr 95% of the lZ5I- of 1251-cu2AP-plasmin and "'I-a2M-plasmin complex reacting equimolar concentrations of active plasmin and "'I-protein- ase inhibitor (0.3 pM) for 15 min at 22 "C. Electrophoresis-Complex formation between '251-plasmin a,AP or a2M was detected by SDS-PAGE on 5 (a2M reactions) or 8% (a2AP reactions) slabs using a Hepes/imidazole (pH 7.4) buffer system (23). The samples were not treated with reductant prior to electrophoresis. Gels were stained, dried, autoradiographed, and cut into 3-mm slices. The radioactivity in each slice was determined in a y counter. a2AP-plasmin and a2M-plasmin complexes were clearly resolved from unreacted proteinase. When plasmin reacts with a2M, 96-98% of the proteinase is covalently bound as determined using this SDS-PAGE system (24). Cell Culture-Rat C6 glioma cells were cultured in minimum es- sential medium (GIBCO) with 10% fetal described (19). Experiments were performed using 90-95% confluent monolayers in 24-well cell culture plates (GIBCO). HUVECs were obtained from American Tissue Type Collection (Rockville, MD) and cultured in 24-well collagen-coated plates (45 pg of collagen/well). The medium was F12-K supplemented with heparin (100 pg/ml), fetal calf serum (20%), and endothelial (75 kg/ml). Cells were passaged two to four times and then brought to near confluence (90-95%) before performing experiments. Plasmin Receptor Binding Studies-Cultures HUVECs were washed three times with Earle's balanced salt (EBSS) containing 10 mM Hepes, pH 7.4, and 10 mg/ml bovine serum albumin. A standard concentration of "'I-DIP-plasmin (0.1 PM) was incubated with increasing amounts of unlabeled DIP-plasmin for 4 h at 4 "C. The medium was then aspirated and the wells were washed three times with EBSS, 10 mM Hepes, pH 7.4. Cell-associated radio- activity was determined after dissolving the contents of the wells with 0.1 M NaOH, 2% SDS. Cellular protein was determined by the method of Lowry, as reviewed by Peterson (25). Specific binding was deter- mined as the difference between radioligand bound in the presence and absence of a 100-fold molar excess of unlabeled DIP-plasmin. Binding isotherms were fitted to the equation hyperbola by nonlinear regression, as previously described (13). The same data were also plotted using the Scatchard transformation. The KT, and B,,, were determined by linear regression. Radioligand Dissociation Rate Determinations-Cultures or HUVECs were incubated with 0.1 WM '2'II-plasmin, 1251-plasmino- gen, 1251-DIP-plasmin or aprotinin-inactivated '2'II-plasmin in EBSS, 10 mM Hepes, 10 mg/ml bovine serum albumin for 2 h at 4 "C. The cells were washed three times, and fresh medium with or without 0.3 FM nonradiolabeled DIP-plasmin (no radioligand) was added to each well (250 $1). The DIP-plasmin represented a 150-fold molar excess to the amount of bound radioligand. With this concentration of DIP- plasmin, the maximum level of binding achievable at equilibrium is less than 2.0% of the total. Therefore, in the presence of the nonra- diolabeled DIP-plasmin, radioligand reassociation following dissocia- tion should be insignificant. After adding the fresh medium to the wells, incubations were conducted for up to 900 s at 4 ', 22 ', and 37 "C. The medium and cells were then separated, and the radioactivity in each mined. Dissociated radioligand (in the medium) was expressed as a percentage of the total radioactivity associated with the cells and medium. In separate studies, the effect of a2AP (0.5 or 2.0 por 4.0 PM) on the dissociation of '*'I-DIP-plasmin was evaluated. All experiments were performed at least in triplicate. Dissociation rate constants ( koff) were determined by linear regression analysis of data obtained during the initial 180 s of each experiment. Correlation coefficients were greater than 0.92. Plasmin Inhibition Studies-C6 cells or HUVECs were incubated with 0.1 p~ 12511-plasmin for 2 h. After washing medium containing a2AP (0.04-2.0 PM) and/or a2M (0.4-4.0 pM) was added back to each well. The molar ratio of proteinase inhibitor in solution to receptor-bound '*'I-plasmin was 20:l or greater. Incuba- tions were conducted for various periods of time at 4 ", 22 PNPGB (0.1 mM) was then added to each well to inactivate the plasmin, followed by SDS-PAGE sample buffer. Reaction products were analyzed by SDS-PAGE. PNPGB rapidly inactivated both free and receptor-bound plasmin. No reaction of receptor-bound I2'I- plasmin with a,AP or a2M was detected when these proteinase inhibitors were added simultaneously with the PNPGB. In order to determine the amount plasmin available for reaction in each study, with 12'II-plasmin for 2 h, washed, and then incubated with 10 mM c- were formed by and excess calf serum as previously cell growth factor of C6 glioma cells or solution for a rectangular of C6 cells was deter- ~) and a2M (1.0 the cells, 250 pl of or 37 "C. of active receptor-bound 12'1- cells were incubated
Page 3
Inhibition of Receptor-bound Plasmin 12331 amino-n-caproic acid (EACA) together with azAP (2.0 WM) or n2M (4.0 p ~ ) . After 60 min at 4 ' or 22 "C, the products were analyzed by SDS-PAGE. EACA displaces plasmin from cellular binding sites (12, 26, 27), allowing rapid reaction with proteinase inhibitors in the solution phase. The rate of reaction of plasmin with a,AP is decreased by EACA; however, the second order rate constant remains above lo5 M" s-l at 22 "C (28). The reaction of cu2M with plasmin is not affected hy EACA (16). In our experiments, essentially plasmin was displaced into solution by the EACA in 60 min. Equiv- alent amounts of "'I-plasmin-inhibitor complex incubating the cells with EACA and a2AP or EACA and a2M. Tem- perature did not affect the maximum yield of '2sIIplasmin-inhibitor complex; nor did extending the incubation time. In fact, equivalent amounts of "'I-plasmin-inhibitor complex 60-min incubations were conducted without EACA. Based on these studies, receptor-hound "'I-plasmin typically retained greater than 95% of the initial activity (before incubation with cells). Each plasmin inhibition study was standardized by assuming that complex forma- tion was 100% complete after incubation with EACA and a2AP or cvzM for 60 min. In some '"I-plasmin inhibition studies, the media and cells were separated after the PNPGB was added. The two fractions were then denatured and analyzed individually by SDS-PAGE. All plasmin inhibition experiments were performed at least twice with duplicate determinations in each study. The results of the studies were aver- aged. Plasmin Inhibition Rate Constants-We propose which is initially receptor-bound reacts with proteinase inhibitors two distinct pathways. In pathway 1, the plasmin (Pm) first disso- ciates from the receptor and then reacts with a2AP or solution as shown by the following equation. 100% of the active were detected after were observed when the that plasmin via cu2M (I) in Pm-receptor -+ Pmm, + Z -+ Pm-Z complex (1) k' is the pseudo-first order rate constant for the reaction of plasmin with a large molar excess of a,AP or n,M in solution. If k' >> kofT, as in the studies presented here, plasmin-receptor dissociation is rate- limiting, and the formation of SDS-stable-a2AP-plasmin complex is a first order process. As will be demonstrated below, reassociation of PmfrVr with receptors is slow compared with the reaction of Pmfr,, with proteinase inhibitors. Therefore, in the presence anM, significant plasmin reassociation is not expected. In pathway 2, receptor-bound plasmin reacts with inhibitor added to the medium. The reaction occurs on the cell surface as shown in the following equation. of a,AP or excess proteinase k r r k' cuk' Pm-receptor + Z -+ Pm-receptor-Zcomplex + Pm-Z complex (2) Unlike pathway 1, the rate of plasmin inhibition depends on the concentration of proteinase inhibitor. The pseudo- first order rate constant for the reaction of inhibitor with plasmin in solution (k') is decreased by the factor LY when the plasmin is receptor- bound. Since the medium and cells were not separated before analysis by SDS-PAGE, Pm-receptor-I complex and Pm-I complex were in- distinguishable. In order to complete our calculations, we assumed that the concentration of proteinase inhibitor in the microenviron- ment adjacent to the cell surface is equivalent to the concentration in the solution as a whole. Since the validity of this assumption was not tested, the effect of the cell surface on the composition of the adjacent solution should be recognized as a possible explanation for our results. Plasmin inhibition data were analyzed according to the following equation. via pathway 2 log[Pm,]/[Pm,,] = k,,,t/-2.303 (3) [Pm,] is the concentration of active plasmin at time t and [Pm,] is the concentration of active plasmin at time 0. The k,,, is an experi- mental rate constant hypothetically equivalent to the sum order (or pseudo-first order) rate constants (k<,tt + ak'). Each k,,, value was determined from linear regression analysis of data generated during the first 180 s of reaction. Correla- tion coefficients ranged from 0.93-0.99. of the first from pathways 1 and 2 RESULTS Plasmin Binding to C6 Cells-High concentrations of active plasmin may create new plasmin(ogen) cell surface binding sites (30). Therefore, equilibrium binding studies were per- formed using radioiodinated DIP-plasmin. Specific binding represented greater than 70% of total binding and was appar- ently saturable (Fig. 1). Scatchard analysis suggested a single class of binding sites with a Ku of 0.9 p~ and a B,,, of 2.6 x 10' sites/cell. The B,,, is in good agreement with the value determined previously for Glul-plasminogen in the same cell system (19). DIP-Plasmin appears to have a slightly higher affinity than Glul-plasminogen (1.7 p ~ ) . binding parameters determined with C6 cells are consistent with those reported for U937 cells and endothelium (11,26). 1251-azAP or "'I-aZM was incubated with C6 cells for up to 2 h at 4 "C. Specific binding, as defined by displacement using a 100-fold molar excess of unlabeled ligand, was not observed with either radioligand. In the concentration range studied (0.04-4.0 pM for a2AP and 0.02-2.0 pM for a2M), nonspecific binding represented less than 0.1% of the total asAP present and 0.4% of the total a2M present. 1251-a2AP-plasmin and 1251-azM-plasmin were incubated with C6 cells for 2 h at 4 "C. The concentration of radioligand was 50 nM; unlabeled ligand was included at variable concen- trations from 0-2.0 p~. Specific binding of the preformed I2'I- proteinase inhibitor-plasmin complexes, as defined by a 40- fold molar excess of unlabeled ligand, Nonspecific binding was unchanged compared with lzsI-azAP and "'1-a2M before reaction with plasmin. By SDS-PAGE and autoradiography, 1251-proteinase inhibitor-plasmin com- plexes present in the medium after 2 h were not digested or altered by the cells. Plasmin-Receptor Complex Dissociation-Incubation cells with 0.1 p~ active plasmin for 2 h at 4 "C did not cause cell detachment (based on cell counts of supernatants), de- crease cell viability (determined by trypan blue exclusion), or change cell morphology (phase-contrast microscopy). The amount of bound "'I-plasmin was 150-250 fmol, which rep- resented less than 2.0% of the total radioligand. Fig. 2 shows the rate of '2sII-plasmin dissociation from the C6 cells at 4 "C. The kOff was 4.0 X lo-* s-' in the absence of DIP-plasmin, as determined from the initial 180 s of analysis. After 15 min, greater than 85% of the radioligand remained bound to the cells. Similar results were observed with '2'I-plasminogen, 1251- DIP-plasmin, and aprotinin-inactivated 12sI-plasmin. I-plasmin dissociation experiments were performed in the presence of 0.3 pM DIP-plasmin in order to prevent reassocia- tion of radioligand. The kOff was increased 2.4-fold to 9.7 X The DIP-plasmin was not observed. of C6 125 If 0 OY 0 1 2 3 4 5 [1251-DIP-PLASMIN] IuMI FIG. 1. Specific binding of DIP-plasmin to C6 cells. Mono- layers of C6 cells were incubated with '"I-DIP-plasmin (0.1 pdifferent concentrations of unlabeled DIP-plasmin at 4 "C for 4 h. After washing, the cells were lysed and specific binding was deter- mined. The inset shows the Scatchard transformation of the binding isotherm. The x- and y-intercepts were 25.4 X lo5 molecules/cell and 23.4 X 10" (molecules/cell) X WM", respectively. ~) and
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12332 Inhibition of Receptor-bound Plasmin 101 I V 0 200 400 Time (sec) 600 800 1000 126 FIG. 2. Dissociation of '251-plasmin, aprotinin-inactivated I-plasmin, and '261-plasminogen from C6 cell receptors. Cells were incubated with 0.1 FM 1251-plasmin (0), aprotinin-inacti- vated "'I-plasmin (A), or '251-plasminogen (0) for 2 h at 4 "C. The monolayers were washed, and fresh medium without radioligand was added. In one set of experiments, cells were incubated with lZsI- plasmin, washed, and then incubated with fresh medium containing 0.3 pM DIP-plasmin (0). At various times, the medium and cells were separated and the radioactivity in each was determined. Dissociated radioligand was calculated as a percentage of that present in the medium and cell-associated fractions. The presented results represent the average of four determinations. TABLE I Effect of a A P and a2M on the dissociation of receptor-bound '2sZ-DZP-plasmin in C6 cells Solution phase x k,, S " 5.1" 5.5 6.5 6.1 5.4 Medium alone Medium + 0.5 p~ azAP Medium + 2.0 p~ a2AP Medium + 1.0 PM a2M Medium + 4.0 p~ a2M I ' Rate constants were determined from an analysis of the initial 180 s. All studies were performed at 4 "C. s-'. If the DIP-plasmin promoted dissociation of recep- tor-bound '"I-plasmin in addition to preventing reassociation, then the true kOff is intermediate between the two determined values (4.0 X and 9.7 X s-') (see "Discussion"). The kOff determined in the presence of DIP-plasmin and the KO presented above were utilized to calculate an approximate association rate constant (ken) of 1.1 X lo3 M" s-l, based on the expression k,, = koff/KD. '251-DIP-plasmin dissociation experiments were performed in the presence of a2AP or a,M. As shown in Table I, neither proteinase inhibitor affected the dissociation of the inactive plasmin. Inhibition of C6 Cell-bound Plasmin by aAP-The reaction of a2AP (0.04 pM) with plasmin (2.0 nM) in solution was studied using the SDS-PAGE method (Fig. 3). As expected, greater than 80% of the active plasmin was bound to a2AP within 60 s at 4 "C. The k,,, was 0.2 s-'. Assuming a linear relationship between [a2AP] and kapp, the second order rate constant was 5.0 X lo6 M-' s-'. This constant is slightly lower than published values (15,31). The difference may reflect the lower temperature used in this study method which is not highly accurate with extremely fast reactions. The reaction of C6 cell-associated '"I-plasmin with a2AP at 4 "C was extremely slow compared with the solution-phase or the SDS-PAGE 100 . . . . ..... . . . . . . . . . . . . . . . . .... .. - x - 50 z z m Q _J a W > H H A 0 200 400 Time (sec) 600 800 1000 FIG. 3. Inhibition of C6 cell-associated plasmin by a2AP. Receptor-bound '251-plasmin was incubated with the following con- centrations of a2AP at 4 "c: 0.04 pM (o), p~ (O), and 2.0 p~ (0). At various times, the reactions were stopped by adding PNPGB (0.1 mM) and SDS-PAGE sample buffer. a2AP- plasmin complex formation was detected by SDS-PAGE. The amount of active (uncomplexed) plasmin was expressed as a percentage of the total plasmin present (active plasmin and a2AP-plasmin For comparison, the reaction of solution-phase plasmin (2.0 nM) with 40 nM a2AP (e) at 4 "C was studied using the same SDS-PAGE procedure. The 1251-plasmin dissociation curves determined in the presence (- - -) and absence (. . . .) of 0.3 p~ DIP-plasmin are repro- duced from Fig. 2 for comparison. 0.1 pM (G), 0.4 pM (A), 1 . 0 complex). TABLE I1 Rate constants for the reaction of C6 cell receptor-bound Plasmin with a 4 P and a2M at 4 "C Inhibitor concentration lo-' x kapD a9AP a?M P M 0.04 0.1 0.4 1.0 2.0 4.0 S " 7.7 12.9 18.7 S " ND" ND 6.1 22.9 7.2 10.2 13.8 37.3 ND ND, not determined. reaction. The k,,, values were significantly lower than the constant determined with plasmin in solution (Table Since the cells and media were denatured and analyzed to- gether in these experiments, the complex formation did not result from selective distribution of the complex into one of the two phases. As described above, plasmin that is initially receptor-bound can react with proteinase inhibitors via two separate path- ways, one that requires plasmin dissociation (pathway 1) and one that does not (pathway 2). For the reaction of C6 cell- associated plasmin with a2AP, the function of proteinase inhibitor concentration. In addition, all of the k,,, values, except for the constant determined with 0.04 FM a2AP, were greater than the koff determined in the presence of DIP-plasmin (the k,,, for 0.04 pM a2AP was still greater than the koff determined without DIP-plasmin). These results strongly suggest that both pathways to the formation of a2AP-plasmin complex when receptor- bound plasmin is reacted with a2AP. The amount plasmin complex formed via pathway 2 is a function of a2AP concentration. Even when the two contributing pathways were considered together (by the k,,), the rate of complex formation between a2AP and receptor-bound plasmin 11). decrease in azAP-plasmin k,,, values varied as a 1 and 2 contribute of a2AP- was
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Inhibition of Receptor-bound Plasmin 12333 260-2,600-fold slower than the rate with both reactants in curves deviated from linearity when time points after 180 s were considered. Possible explanations for this are discussed below. Inhibition of C6 Cell-associated P h m i n by aZM-As deter- mined by the SDS-PAGE method, the k,,, for the reaction of solution-phase plasmin (2.0 nM) with 0.4 p~ a2M at 4 "c was 0.16 s-'. The corresponding second order rate constant was 4.0 x lo5 M-' s-' (assuming a linear relationship between k,,, and [azM]). The reaction of receptor-bound plasmin with a2M at 4 "C was extremely slow (Fig. 4). Only the two highest a2M concentrations yielded ka,, values that exceeded the koff determined in the presence of DIP-plasmin. The k,,, values did show a weak dependency on a2M concentration, varying from 6.1 X s-' with 0.4 pM aqM to 1.4 X p~ a2M (Table 11). These studies demonstrate that receptor- bound plasmin is highly protected from inhibition by a2M as well as a2AP. While direct reaction bound plasmin may have occurred at a slow rate, the fraction of a2M-plasmin complex formed via pathway 2, as opposed to pathway 1, was small compared with aZAP. Inhibition of Plasmin by Premixed Solutions of a A P and a2M-The k,,, values presented in Table I1 suggest that a2AP is a more efficient antiplasmin than a2M independent of whether the plasmin is receptor-bound or confirm this hypothesis, C6 cell-associated '251-plasmin was reacted with premixed solutions of azAP (0.5 p ~ ) (1.4 p~) at 4 "C (Table 111). After incubation for 2.5 min or 15 min, the ratio of a2AP-plasmin to a2M-plasmin was ap- proximately 4:l. The combination of azAP and a2M bound more '2511-plasmin than either proteinase inhibitor porting the role of pathway 2 as a contributor to the inhibition of plasmin in these experiments. When the '2sI-plasmin and the proteinase inhibitors were all present in solution, the ratio of a2AP-plasmin to a2M-plasmin suggest that azAP and a2M participate regulation of solution-phase and receptor-bound plasmin (by all relevant pathways). Effect of Temperature on the Regulation of Receptor-Bound of complex formation solution. Some plasmin inhibition s" with 4.0 of a2M with receptor- in solution. To and aqM alone, sup- was again 4:l. These studies comparably in the Z a W > H I- u 4 T i m e (secl FIG. 4. Inhibition of C6 cell-associated plasmin by azM. Receptor-bound "'I-plasmin was incubated with the following con- centrations of anM at 4 "C: 0.4 pM (0), 1.0 p~ (o), 2.0 pM (A), and 4.0 pM (0). Reaction products were analyzed by comparison, the reaction of solution-phase plasmin (2.0 nM) with 0.4 p M a2M (e) at 4 "C was studied using the same procedure. The lZ5I- plasmin dissociation curves determined with (. . . .) of 0.3 p~ DIP-plasmin are reproduced from Fig. 2 for compar- ison. SDS-PAGE. For (-- -) and without TABLE 111 Reaction of C6 cell-associated and solution-phase plasmin with premixed solutions of a2AP and apM 12,51-Plasmin in solution was incubated with a premixed combina- tion of a&' phase). Complex formation with the two proteinase inhibitors was determined by SDS-PAGE as described under "Experimental Proce- dures." Receptor-bound plasmin was generated by incubating C6 cells with 0.1 p~ '2sII-plasmin for 2 h at 4 "C. The cells were washed, and the same mixture of a2AP and a2M was added to the wells for the indicated times. Total plasmin reacted mirz % Solution-phase 15 100 Receptor-bound 2.5 15.6 f 5.3" ReceDtor-bound 15 72.6 f 6.6 (0.5 pM) and anM (1.4 FM) for 15 min at 4 "c (solution Plasmin species Time complex ' % of total 18 15.2 f 4.1 84.8 22.0 f 2.4 78.0 012M apAP complex % of total 82 f 4.1 f 2.4 Mean * S.E. \ """_ .... .. A 0 60 120 180 0 60 120 IS0 T i m e (sec) FIG. 5. Effect of temperature on the inhibition of receptor- bound plasmin by a,AP. C6 cell-associated "'I-plasmin was reacted with 0.5 pM (0) or 2.0 p M a2AP (A) at 22 "c (panel A ) or 37 "c (panel B). Reaction products were analyzed using the SDS-PAGE method. 12'I-plasmin dissociation was studied at the same tempera- tures in the presence plasmin. (- - -) and absence (. . . .) of 0.3 p~ DIP- Plasmin-The formed at 4 "C in order to minimize plasmin dissociation and to limit the effect of plasmin on cellular structure. In order to confirm the results of the 4 "C studies, additional experiments were performed at 22 and 37 "C. In these experiments, lz5I- plasmin was incubated with C6 cells for 2 h at 4 "C. The cells were then washed and brought to equilibrated medium (with or without proteinase inhibitors). The rate of plasmin dissociation increased as a function of temperature (Fig. 5). In the absence of DIP-plasmin, the koff values were 6.4 X s-' and 1.2 X respectively. In the presence of DIP-plasmin, the kOff values were 1.4 x s" and 2.3 x respectively. The rate of reaction of C6 cell-associated plasmin with a2AP was also increased significantly at 22 and 37 "C (Table IV). The k,,, derived at 37 "C with 2.0 p~ a2AP was 6.7-fold higher than the kOff derived at the same temperature with DIP-plasmin. As was the case at 4 "C, k,,, values for the reaction of receptor-bound plasmin with aZM exceeded the k,ff (with DIP-plasmin) only when the proteinase inhibitor concentration was high. The ratio of k,,, to koff with 4.0 p~ a2M was 1.8 at 22 "C and 2.3 at 37 "C. Distribution of aAP-Plasmin After Inhibition of Receptor- initial plasmin inhibition studies were per- 22 or 37 "C with pre- s-l at 22 and 37 "C, s-' at 22 and 37 "C,
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12334 Inhibition of Receptor-bound Plasmin TABLE IV Effect of temperature on the reaction of receptor-bound plasmin with a d P and a2M All experiments were performed using C6 cell cultures. Rate con- stants were determined from an analvsis of the initial 180 s. Rate constant lo-'' x k 22°C 37°C Solution component S" 0.6 1.4 3.6 7.5 1.6 2.5 S" kw None 0.3 FM DIP-plasmin 0.5 FM a,AP 2.0 FM CQAP 1.4 pM a,M 4.0 p M anM 1.2 2.3 k,,,,, k,,,,, ku,, ken,, 8.6 15.3 2.9 5.3 bound Plasmin-In order to determine whether a2AP-plasmin complex remains associated with C6 cells after formation on the cell surface, receptor-bound "'I-plasmin was reacted with 1.0 pM a2AP for various amounts of time at 4 "C before adding PNPGB. The medium and cells were then separated and analyzed individually by SDS-PAGE. Based on the studies presented in Fig. 3, we predicted that more than 50% of the a2AP-plasmin complex was formed via pathway 2; however, only 15% of the azAP-plasmin complex was recovered in the cellular fraction at 120 s. At 5, 10, and 15 min, only 4-10% of the a2AP-plasmin was cell-associated (range of values ob- tained in two experiments; data not shown). Importantly, the cell-associated a,AP-plasmin always represented less than 5% of the cell-associated radioactivity, confirming the report of Miles and Plow (8). These studies suggest that a2AP-plasmin complex is released into solution after formation on the cell surface. By SDS-PAGE and autoradiography, the a2AP-plas- min on the cell surface and in the medium were identical. In order to confirm that a2AP-plasmin complex formed on the cell-surface is dissociated into solution, C6 cell-associated '"I-plasmin was incubated with DIP-plasmin and a2AP si- multaneously at 4 "C. The medium was then separated from the cells and recovery of 1251-plasmin (and/or azAP-1251-plas- min) in the medium was determined (Fig. 6). As expected based on Fig. 2, DIP-plasmin alone increased the recovery of '"I-plasmin in the medium. The combination of aPAP and DIP-plasmin increased recovery of 1251-plasmin in the medium still further, and the effect of the a2AP was concentration- dependent. Since DIP-plasmin alone prevented '251-plasmin reassociation with cellular receptors, the additional '251-plas- min recovered in solution in the presence represented I ) reaction of azAP with receptor-bound "'I- plasmin at the cell surface (pathway 2) and 2) dissociation of a2AP-'"1-plasmin from the cell-surface into the medium. The recovery of a2AP-plasmin in these experiments was slightly lower than that demonstrated in Fig. 3. This difference may reflect, at least in part, a,AP-plasmin complex which formed on the cell surface but had not yet dissociated. Inhibition of Plasmin Bound to Endothelial Cells-In pre- liminary experiments, we demonstrated specific and saturable binding of radioiodinated DIP-plasmin to HUVECs in cul- ture, confirming the work of other investigators (26). A single class of binding sites was detected. The KO was 0.6 p~ and the B,,, was 3.1 X lo7 sites/cell (data not shown). Plasmin-receptor dissociation studies were performed at 4 "C. The was 3.2 X s-' in the absence of DIP-plasmin and 8.9 X s-' in the presence of 0.3 p~ DIP-plasmin. The rate of plasmin inhibition by a2AP (0.5 and 2.0 psignificantly decreased when the plasmin was bound to HU- VECs (Fig. 7). Nevertheless, the k,,, values exceeded the kOff suggesting that pathways 1 and 2 were involved. HUVEC- of a2AP most likely ~) was 2 60 W z z H z 40 H x rn a 2 20 O CON D I r n 0.1 1.0 I DIPA + ON 8.0 (UWI I FIG. 6. Reaction of receptor-bound plasmin with a2AP in the presence of DIP-plasmin. C6 cells were incubated with 0.1 PM "'I-plasmin for 2 h at 4 "C. After washing, the monolayers were incubated with the following solutions for 15 min: fresh medium only (CON), medium containing 0.3 FM DIP-plasmin (DIP-Pm), or me- dium containing 0.3 PM DIP-plasmin and a2AP (DIP-Pm + a d P ) . The concentration of a2AP was varied as shown. After incubation, the medium was recovered from each well without the cells. Total radioactivity in each sample of medium was determined. For samples treated with a2AP, SDS-PAGE was performed to determine the percentage of '2sII-plasmin that was complexed with a2AP (due to either dissociation from the cell surface in an active state or reaction with a2AP on the cell surface). The samples that did not include proteinase inhibitor (CON and DIP-Pm) were treated with 1.0 PM azAP immediately after recovery. Active plasmin was then deter- mined as a2AP-"'I-plasmin complex by SDS-PAGE. W > H k- u 4 : 20. 1 0 ~ ' " " ' 0 60 120 160 Time (sed FIG. 7. Dissociation and inhibition of receptor-bound plas- min in HUVEC cultures. HUVECs were incubated with 0.1 pM '2sI-plasmin for 2 h at 4 "C. The cultures were then washed and dissociation was studied in the presence (- - -) and absence ( . . . . ) of 0.3 PM DIP-plasmin. The washed cultures were also incubated with 0.5 FM a2AP (0) or 2.0 PM a2AP (A) at 4 "C. Reaction was monitored by the SDS-PAGE method. associated plasmin was also protected from inhibition by azM. For the most part, reaction of plasmin that was initially receptor-bound with azM depended on plasmin dissociation; k,,, values were not significantly greater than the k,ff deter- mined with DIP-plasmin (data not shown). These studies demonstrate that dissociation and inhibition of receptor- bound plasmin are comparable in HUVECs and C6 cells; the function of the plasmin receptor, in relation to antiplasmin proteinase inhibitors, is most probably conserved across spe- cies and cell lines. DISCUSSION The structure of plasmin includes five tandem repeat do- mains termed "kringles" followed by a C-terminal "trypsin-
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Inhibition of Receptor-bound Plasmin 12335 like" enzyme domain (32). The kringles mediate noncovalent binding interactions with numerous macromolecules includ- ing fibrin (32), thrombospondin (34), histidine-rich glycopro- tein (35), glycolipids (36), and other incompletely character- ized molecules in cellular membranes and the extracellular matrix (8,37). The abundance and diversity binding molecules suggests that the activity of plasmin may be important in numerous environments. In addition, plasmin demonstrates broad substrate may have deleterious effects when not localized or restricted by specific binding macromolecules. Since a2AP and anM rapidly inactivate plasmin that is not bound to "protective" macromolecules, the activity of plasmin in vivo may be almost entirely attributable to bound forms of the enzyme. The reaction of the plasma proteinase inhibitors with plasmin bound to other macromolecules remains poorly understood. If aOAP is less reactive plasmin, then the importance of other antiplasmin proteinase inhibitors may be relatively enhanced. In addition, since different kringles mediate the noncovalent binding to various macromolecules, it is likely that the proteinase inhibitors will be affected differently by each plasmin binding structure. In this investigation, we studied the inhibition of receptor- bound plasmin by aOAP and a2M. The major conclusions of this study are: 1) receptor-bound plasmin is protected from inhibition by a2M as well as a2AP; 2) in the presence of low concentrations of a2AP, the majority of the plasmin that is initially receptor-bound is inactivated only after dissociation from the cell surface, and this is a relatively slow process; 3) when the concentration of a2AP approximates or exceeds that present in plasma (1.0 PM), receptor-bound plasmin is inac- tivated by direct reaction with a2AP as after dissociation; and 4) the reaction of plasmin that is initially receptor-bound with a2M ways; however, even high concentrations of a2M react mini- mally with plasmin at the cell surface. Rapid reaction of plasmin with azAP depends on the non- covalent association of the kringle-1 domain (Kl) with the C- terminal region of the inhibitor. Since K1 is involved in the plasmin(ogen)-receptor interaction (38), it is decrease in a2AP activity against receptor-bound plasmin reflects the occupied K1 domain. A similar explanation is well accepted for the decreased rate of reaction of a2AP with plasmin bound to fibrin (28). For the reaction of a2M with receptor-bound plasmin, a different explanation is necessary. There is no evidence that the K1 domain plays any role in this reaction (16). In fact, miniplasmin, which lacks the K1 domain, reacts more rapidly with a2M than does plasmin (31, 39). We speculate that steric constraints or diffusional limi- tations restrict the reaction of receptor-bound plasmin with aZM (Mr = 718,000); however, it is interesting that similar constraints apparently do not affect the reaction trypsin bound to sheep red blood cells (18). More studies will be necessary to explain the protection plasmin from a2M. The k,ff values determined in the presence and absence of DIP-plasmin differed by 2.4-fold at 4 "C, 2.2-fold at 22 "C, and 1.9-fold at 37 "C. These modest differences may have resulted entirely from the ability of the DIP-plasmin to pre- vent 1251-plasmin reassociation with receptors; however, it is also possible that DIP-plasmin increases the rate plasmin dissociation. The latter suggestion implies negative cooperativity in the plasmin receptor binding system, which is not supported by our own DIP-plasmin equilibrium binding studies. However, in experiments with low concentrations of of plasmin(ogen) because specificity, this enzyme with bound forms of of plasmin at the cell-surface as well may also involve two path- possible that the of anM with of receptor-bound of '*'I- alAP or a2M, the ka,, values were consistently less than the kOff determined with DIP-plasmin. This should not be possi- ble, based on calculations that consider the rate of plasmin inhibition in solution, the rate receptors, and the number of receptors/cell. We are currently exploring possible explanations for the effect of DIP-plasmin on the kOff. Since we are unable to resolve this issue at the present time, we have chosen to interpret the plasmin inhi- bition data conservatively, using the kOff values determined in the presence of DIP-plasmin to judge whether receptor-bound plasmin is reacting with a2AP or a2M at the cell surface. If indeed cooperativity is affecting our kOff determinations, then the proportion of receptor-bound plasmin reacting via path- way 2 may be higher than suggested. Bauer et al. (40), studied the interaction of human plasmin with mini-pig aortic endothelial reported kOff values of 2.3 x lo-* s" and 1.3 X lo-' s-l at 22 "C in the presence and absence values are approximately 20-fold greater than the k,cf values determined with C6 cells at the same temperature in this investigation. Bauer et al. (40) also reported KD values that were 50-fold lower than those presented here for C6 cells or HUVECs. It is difficult to reconcile these studies without suggesting that different plasmin-receptor sites are available in the mini-pig aortic endothelial HUVECs confirm the work of Miles et al. (8, 26). These investigators were not able to demonstrate a high affinity plasmin binding site in this cell line. While dissociation and inhibition studies in HUVECs and C6 cells yielded equivalent results, it is still possible that some cells express plasmin-binding membrane structures that are ent in function from those reported In some plasmin inhibition experiments, the rate decreased as the incubation was continued past 180 s. The cause of this remains unclear. If there is heterogeneity in the plasmin binding sites or in the 1251-plasmin itself, then the reported k,,, values most closely represent the more rapidly inhibited fractions of receptor-bound plasmin. One possible source for heterogeneity in the 1251-plasmin is carbohydrate structure. Purified human plasminogen preparations consist of two isozymes; however, the KD values for the interaction of these two isozymes with C6 cells are similar (2.2 p~ with plasminogen type I and 1.5 PM with plasminogen type 11). We have not studied the dissociation of purified plasminogen isozymes from C6 cell receptors; however, these small differ- ences in KD may reflect small differences in kOff as well. It is unlikely that a subpopulation of plasmin-receptor complex exists which is completely resistant to inhibition; after a 60- min incubation with a2AP, receptor-bound plasmin pletely inhibited whether or not EACA was included to dis- sociate the plasmin. In addition, plasmin was inactivated within 15 min at 4 "C. A cell-surface receptor that specifically binds uPA (u-PAR) has been purified and cloned (41, 42). plasmin, uPA that is bound to u-PAR reacts plasminogen activator inhibitors (PAI-1 and PAI-2) (43, 44). Ellis et al. (43) reported rate constants of 4.5 x lo6 M" s-' and 3.3 X lo5 M" s" for the reaction u-PAR-bound uPA PAI-1 and PAI-2, respectively. Both of these rate constants were decreased by only 40% compared with the solution- phase reactions. We speculate that the activity complex may be regulated by changes in the local concentra- tions of PAI-1 and PAI-2. By contrast, the plasmin receptor provides a mechanism for the expression of enzyme activity in the presence of consistently high concentrations of a2AP and a2M. of plasmin association with cells. These investigators of unlabeled enzyme. These cells. Our studies with the plasmin differ- here. of reaction was com- nearly 80% of the cell-bound By contrast with readily with with of uPA-u-PAR
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CASTING CALL || Cezar
Cezar is quite the whore, who wrote into us, from sunny San Paulo. Rather the describe to you what makes him a good canidate, we thought we let his application speak for its self. Here's what he wrote.
"I´m 37 years old, from São Paulo, Brazil. I´m applying here, models-europe, because I´m going to try porn life in Europe, so, despite the fact that I´m in Brazil now, soon I´ll be in Paris or London…. Well, I'm a total sex addict. I LOVE MEN, COCKS , CUM AND PISS. LOVE!!! I want to spend my life surrounded by dicks, men…. I want to live for porn, men, sperm……
I love to suck dicks, eat sperm, have my face washed with cum and piss. I love being fucked….sometimes I love to fuck. I love bukkakes!!!! My dream is to do something like "damon blows america" but "LEO BLOWS EUROPE". I want to go from Lisbon to Moscow sucking dicks, thousands of them!!!!!!!!! Being pissed too. Fucked sometimes, gangbanged….
My other fantasy, and I see that lots of people look for it, is I WANT TO BE GANG BANGED BY MIDGETS!!! yes!!! fucked by midgets, and drinks their sperm. I want to suck and want to be fucked by real dad/son. brothers…. all this taboo stuff."
The proceeding are his stats from his application. What do you think, is he worthy of being drafted into Paul Morris' Army of Notorious pig bottoms? Enjoy his application , then go to Facebook and vote.
Cezar's Application. –
- [cock size] => 8″
- [cut/uncut] => uncut
- [height] => 5'10"
- [weight] => 175 lbs
- [hair] => black
- [eye] => brown
- [bodytype] => athletic
- [ethnic] => Brazilian
- [recdrug] => Not opposed to them but dont do them often
- [fucking] =>hungry bottom
- [sucking] => cumslut throat
- [piss play] => yes please
- [nipple play] => vers
- [fleching] => bottom
- [fist fucking] => bottom
- [rimming] => bottom
- [S&M] => bottom
- [turn ons] => I´m a total sex addict. I LOVE MEN, COCKS , CUM AND PISS. LOVE!!! I want to spend my life surrounded by dicks, men…. I want to live for porn, men, sperm……
- [turn offs] => I love a musky man but bad hygiene can be a big turn off for me
- [fanatasy] => I WANT TO BE GANG BANGED BY MIDGETS!!! yes!!! fucked by midgets, and drinks their sperm.
- [sexual talent] => I'm a total fucking whore
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Should Cezar Be Our Next TIMWhore? Click here to vote and help us decide.
Think you have what it takes to be on of Paul Morris' Men? Fill out an application here.