text:
The Fourteenth Century
The papacy had reached the zenith of its political power in Europe under Innocent III. His death in 1216 was followed by a period of eclipse and, finally, catastrophe. The popes continued to struggle for supremacy against Germany's "Holy Roman" …Read More
For the Love of God and Man
When Paul called the elders of Ephesus to meet him at Miletus for a final opportunity of fellowship and instruction, he warned them of serious problems that would emerge in the church. "I know," he said, "that after my departure fierce …Read More
The Pagan Agenda of the Code
As I lecture around the world on the Da Vinci Code, a typical response is to tell me to relax, enjoy the book as a fun read of fiction, and move on. Let me say that it is indeed a …Read More
Truly Reformed
Reformed theology has an image problem among the ranks of evangelical Christianity. And anyone who has had the unfortunate problem of being either misunderstood or misrepresented knows that it is not an easy task to repair one's image. Other articles …Read More
Word and Sacrament in Worship
In those fundamentalist churches in which I was raised, most Sunday mornings the minister preached from a well-worn Bible, told a few stories to illustrate his point and then reminded us that Jesus is our only hope of heaven. But …Read More
The Meal That Divides
On the night of the institution of the Lord's Supper, Jesus prayed for His disciples and for those who would become His disciples, "that they may all be one" (John 17:21). This was a profound prayer for unity among Christ's …Read More
Our Bloody Religion
It has been said that Christianity is a bloody religion. This reference is usually made by critics who point to the wars, inquisitions, trials, and executions carried out over the years in the name of Christianity. While we must admit …Read More
Creator and Creature
God's Word starts with the creation by God of all things out of nothing. Genesis chapters 1 and 2 present creation as the foundation of all scriptural truth. The first eleven chapters of Genesis are foundational to the entire New …Read More
They Devoted Themselves to Prayer
From the Mount of Olives, where Jesus ascended to heaven, the disciples went to a prayer meeting. They met in a familiar upstairs room in Jerusalem. All 11 apostles and the women who had been with Jesus were there. They …Read More
Family vs. Culture
The family is the foundation of culture. This is not a bromide of the Christian right, but plain fact, as every anthropologist will tell you. Families associate with groups of families, forming networks of social interdependence as families make a …Read More
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text:
- Features
- OPTISIM Burner Provides Excellent Simmering as Low as a 400 BTU Effect. Perfect for the Most Delicate Liquids and Sauces
- Deep Recessed Maintop Captures Boil Overs and Spills for Easy Clean-up
- Automatic Re-ignition
- Centralized Front Controls for Easy Use
- Low-profile Continuous Grates Provide a Versatile Platform to Work on, Optimizing use of Various Burner Sizes
- Newly Designed Maintop Conforms to the Bosch Family Design
Overall Dimensions | |
Width: | 37" |
Depth: | 21 1/4" |
Height: | 3 13/16" |
Cut Out Dimensions | |
Width: | 34 15/16" |
Depth: | 19 1/8" |
Height: | 3 13/16" |
Burner BTU (min/max) | |
Left Front Burner: | 12,000 BTU |
Left Rear Burner: | 12,000 BTU |
Center Burner: | 16,000 BTU |
Right Rear Burner: | 5,500 BTU |
Right Front Burner: | 12,000 BTU |
Total BTUs Natural Gas: | 57,500 |
Total BTUs LP: | 47,800 |
Miscellaneous | |
Power Required: | 120 Volts, 3 Wire 60Hz Power Cord |
Amps: | 15 |
Installable above Oven: | Yes |
Sealed Burners: | Yes |
Automatic Re-Ignition: | Yes |
POWERSIM Burner: | N/A |
OPTISIM Burner: | Left Front, Right Rear |
Controls: | Front Controls with Red Indicator Light |
Knob Material: | Plastic Knobs |
Knob Color: | White with White Bezel |
Accessories | |
NLP Conversion Kit: | NEZ1054 |
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subset: commoncrawl


text:
The Middleby Corporation Reports Third Quarter Results
The Middleby Corporation (NASDAQ: MIDD), a leading worldwide manufacturer of restaurant and foodservice cooking equipment, today reported net sales and earnings for the third quarter ended October 1, 2011. Net earnings for the third quarter were $23,461,000 or $1.26 per share on net sales of $218,720,000 as compared to the prior year third quarter net earnings of $20,602,000 or $1.13 per share on net sales of $177,793,000.
2011 Third Quarter Financial Highlights
- The third quarter financial statements include the results of the recently completed acquisitions of Danfotech Inc. ("Danfotech"), a manufacturer of meat presses and defrosting equipment for the food processing industry acquired on July 5, 2011, Maurer-Atmos ("Maurer"), a manufacturer of batch and continuous ovens for the food processing industry acquired on July 22, 2011 and Auto-Bake Proprietary Limited ("Auto-Bake"), a manufacturer of automated baking ovens for the food processing industry acquired on August 1, 2011. Results from these recent acquisitions reduced net earnings by $0.8 million or $0.04 per share during the quarter.
- Net sales increased 23.0% in the third quarter. Excluding the impact of acquisitions, sales increased 5.9% during the third quarter. This increase included a 10.1% sales increase at the Commercial Foodservice Equipment Group and a 25.6% sales decrease at the Food Processing Equipment Group as compared to the prior year quarter. Although revenues for the Food Processing Equipment Group declined during the quarter, the incoming order rate and backlog increased.
- Gross profit increased to $87.3 million from $70.7 million. The gross margin rate improved to 39.9% from 39.8%. The improvement in the gross margin rate reflects efficiency gains from the consolidation of production facilities and other integration initiatives, offset by lower margins at newly acquired companies and the impact of sales mix.
- Operating income increased 16.2% to $37.2 million from $32.0 million on higher revenues. Operating income reflected the dilutive effect of acquisitions completed in the third quarter as these operations were reorganized and integrated within Middleby operations.
- Non-cash expenses recorded during the third quarter included $5.3 million of depreciation and amortization as compared to $3.8 million in the prior year third quarter. Non-cash share based compensation expense increased to $5.5 million in the 2011 third quarter as compared to $3.7 million in the 2010 third quarter.
- Provisions for income taxes increased to $11.8 million at a 34% effective rate in comparison to $9.4 million at a 31% effective rate in the prior year quarter. The current year effective rate reflects favorable adjustments for increased tax deductions. The prior year period effective rate reflects a non-recurring benefit to tax reserves.
- Total debt at the end of the 2011 third quarter amounted to $303.6 million as compared to $243.6 million at the end of the third quarter 2010. The increase in debt reflects the funding of $118.6 million related to the acquisitions of Beech, Lincat, Danfotech and Auto-Bake during the first nine months of fiscal 2011. During the third quarter the company also repurchased $3.5 million of Middleby common shares of stock at an average price of $69.10 per share.
Selim A. Bassoul Chairman and Chief Executive Officer said, "At our Commercial Foodservice Equipment Group, industry conditions remained positive during the quarter and we realized continuing revenue gains resulting from growth in international business and with our chain customers. We continue to develop new business with restaurant chain customers as they look to utilize our cooking technologies to lower their operating costs and improve the efficiency of their restaurant operations.
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text:
Awesome organic tacos, but where's my beer?
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text:
Toe Rail Replacement with White Oak or ?
View Single Post
Laugther is Good Medicine
Join Date: Oct 2012
Location: Bonifay, FL 32425 & Huntingdon, Pa 16652
Posts: 146
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Toe Rail Replacement with White Oak or ?
I have evaluated my Toe Rail and it's fairly soft, (42 yr's. old) cracked and may not last me through my intended life of the boat for me (3 to 4 yr's.).
I measured the side toe rails @ 2 7/8" high, 2 1/2" at base, 2 1/4" at top (tapered). I need 36' feet for one side X's 2 = 72' feet total, bow toe rails a little different about 6' feet and curved stern rails = 4' feet.
I have received recommendations for: White Oak or Black Locust for internal structure members, that are sealed with epoxy to protect them.
I priced the Teak out and it will run about $ 1.5 K to $ 2.0 K bucks for all of it. I planed on re-using most of the S.S. hardware but will add $ 300.00 more for replacment hardware as needed.
My No# 1 Question, has anyone used White Oak (epoxy sealed) for Toe Rails
and what were your results ______________ ?
I can buy the Teak ($ 2K) but would rather use USA Wood ($ 300.00). I do not want to go through all this work and have So, So Results. I don't want the boat to look like a 2nd hand ... repair deal, and distract from the boats appearance & resale. I have all the necessary shop tooling to reproduce this (replacement) toe rail with 99% reproduction results.
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text:
See items selected especially for you. Shop recommendations.
Exclusive finds with a new theme each month.
Shop: Pop-In
Our director of creative projects shares what she's into.
Read More
Stay updated with a steady stream of new info.
An elegant neckline is the focal point of a soft-knit staple available in a wardrobe's worth of colors.


text:
Subd Lots
Tourist Spot
About Us
Join us
Cebu Maps
Questions & Answers
Sell Your home
Latest on Pag-ibig
Does your agent know how to interpret contracts?
Raw Land Sale with Sea View P500/sqm San Fernando Cebu
Raw Land Sale Mandaue City
How to be a millionaire in 6 years
House & Lot Gallery
Condominiums Gallery
Rawland, beach lot, etc.
How to purchase online
El Montegrande extension is the latest product offering by the developer. The facilities include: concrete roads, electrical system, water system. Price: P4,000 per square meter Reservation: P5,000 Down Payment: P15% Balance of 85% payable in 5 years no interest Please click the thumbnail map at the right to enlarge
Related Searches: Cebu lot for sale, liloan lot for sale, liloan cebu lot for sale, lot properties cebu liloan, cebu lot, el montegrande heights liloan cebu, lot only for sale in liloan
was founded by dynamic and illustrious men of great foresight in addressing community concerns, like the ever increasing problems on housing in urban areas
Circa 1996, AVELINO S. ALVENIZ, who was still struggling to earn a college degree, started acquiring lots for Pag-ibig members which he would develop as housing areas on individual basis. He did all the marketing and processing of Pag-ibig Housing Applications, including the release of funds and the completion of the houses. So that from 1996 to year 2002, he was able to develop two (250) lots and constructed houses thereon for the Pag-ibig members/clients.
With such experience behind, he put up and founded "PSALM PROPERTY VENTURE AND DEVELOPMENT", a sole proprietorship, under the name of AVELINO S. ALVENIZ. The firm undertook the following subdivision/housing development projects, to wit:
With the foregoing projects, the firm has acquired some fixed assets, such as heavy equipment, i.e., Buldozers, Graders, Pisons, Dumptrucks, Rollers and similar other machineries through sale or lease, and some units of vehicles, and other necessary tools. It has employed a sizeable number of employees – field and office – and has acquired untarnished good name and goodwill.
The members and officers of the corporation have attained skills and experience behind them and such that PSALM PROPERTY VENTURE AND DEVELOPMENT CORPORATION, if given more time and assistance could still be enhanced and become a notable enitity in the housing industry.
El Monte Grande is on going in construction. But lots are offered for pre-selling now and if you see the site, it is fast progressing into a fully developed subdivision. You now have the opportunity to invest in your own lot at the lowest price possible at the location that you like best.
El Montegrande Extension 1
El Montegrande Subdivision (Extension 2)
Click the thumbnail below to enlarge
to Gallery


text: station. Fort Hood was my first, and I've been here six years. Now it's time for a change in scenery and our kids will get to see a different place." The assignment to Hawaii was somewhat of a deciding factor for the Scotts. They probably would have re-enlisted regardless, but being stationed in Hawaii makes it a sweeter deal, said Derek. The Scotts both agree that the assignment to Hawaii is a high priority on their list of things to do. Because of this, they both are making special preparations. "I'm still practicing my beach walk," said Derek, a native of Newport News, Va. "I will have it perfected by the time we reach Hawaii." "I've been working on my abs," said Dawana, a native of Rocky Mount, N.C. The Scotts are scheduled to report to Hawaii in March.


text:
This Internet-Draft is no longer active. A copy of the expired Internet-Draft can be found here: http://www.ietf.org/archive/id/draft-begen-avtcore-rtp-duplication-01.txt
Packet loss is undesirable for real-time multimedia sessions, but can occur due to congestion, or other unplanned network outages. This is especially true for IP multicast networks, where packet loss patterns can vary greatly between receivers. One technique that can be used to recover from packet loss without incurring unbounded delay for all the receivers is to duplicate the packets and send them in separate redundant streams. This document explains how Real-time Transport Protocol (RTP) streams can be duplicated without breaking RTP media streams, or RTP Control Protocol (RTCP) rules.
Ali Begen <email@example.com> Colin Perkins <email@example.com>
(Note: The e-mail addresses provided for the authors of this Internet-Draft may no longer be valid)


text:
Molecular biology of ADAMTS13 and diagnostic utility of ADAMTS13 proteolytic activity and inhibitor assays.
ABSTRACT ADAMTS13, a reprolysin-like metalloprotease, limits platelet-rich thrombus formation in the small arteries by cleaving von Willebrand factor (vWF) at the Tyr1605-Met1606 peptide bond. Deficiency of plasma ADAMTS13 activity, due to either an inherited or an acquired etiology, may lead to a potentially lethal syndrome, thrombotic thrombocytopenic purpura (TTP). Molecular cloning and characterization of the ADAMTS13 gene have provided further insight into the structure-function relationships, biosynthesis, and regulation of the ADAMTS13 protease, in addition to understanding the pathogenesis of TTP and perhaps other thrombotic disorders. ADAMTS13 consists of a short propeptide, a typical reprolysin-like metalloprotease domain, followed by a disintegrin-like domain, first thrombospondin type 1 (TSP1) repeat, Cys-rich domain, and spacer domain. The carboxyl terminus of ADAMTS13 has seven more TSP1 repeats and two CUB domains. ADAMTS13 is synthesized mainly in hepatic stellate cells, but also in vascular endothelial cells. Recognition and cleavage of vWF require the proximal carboxyl terminal domains, but not the middle and distal carboxyl terminal domains. Cleavage of vWF appears to be modulated by shear force, binding to platelet or platelet glycoprotein-1balpha, heparin, inflammatory cytokine (interleukin-6), and chloride ion. At the site of thrombus formation, the ADAMTS13 may be inactivated by thrombin, plasmin, and factor Xa. Having a sensitive and specific assay for ADAMTS13 activity is not only critical to understand the basic biology of ADAMTS13 protease, but also to facilitate a more timely and accurate clinical diagnosis of TTP, and to initiate potentially life-saving plasma exchange therapy. Although many assays have been developed and tested for clinical applications, the fluorescent resonance energy transfer-vWF73 assay appears to be the simplest and most promising assay to date.
- [Show abstract] [Hide abstract]
ABSTRACT: We consider the propagation of few-cycle pulses (FCPs) in cubic nonlinear media exhibiting a "crystal-like" structure, beyond the slowly varying envelope approximation, taking into account the wave polarization. By using the reductive perturbation method we derive from the Maxwell–Bloch–Heisenberg equations, in the long-wave-approximation regime, a non-integrable complex modified Korteweg-de Vries equation describing the propagation of circularly polarized (CP) FCPs. By direct numerical simulations of the governing nonlinear partial differential equation we get robust CP FCPs and we show that the unstable ones decays into linearly polarized half-cycle pulses, whose polarization direction slowly rotates around the propagation axis.Optics Communications 02/2012; · 1.54 Impact Factor -
Article: Current status in diagnosis and treatment of hereditary thrombotic thrombocytopenic purpura.
Hereditary genetics : current research. 01/2014; 3(1). - Moritz Lambers, Neil A Goldenberg, Gili Kenet, Fenella J Kirkham, Daniela Manner, Timothy Bernard, Rolf M Mesters, Ralf Junker, Monika Stoll, Ulrike Nowak-Göttl[Show abstract] [Hide abstract]
ABSTRACT: OBJECTIVE: Previous studies in adults and mice have implicated ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), also known as von Willebrand factor (VWF)-cleaving protease, as a protective factor for stroke. Here we investigated ADAMTS13 in 208 pediatric patients with arterial ischemic stroke (AIS) and 125 population-based control children in a frequency-matched case-control study. METHODS: The proportion of patients/controls with ADAMTS13 activity levels below and above the 10th percentile was compared. Additionally, in a quintile comparison, the proportion of patients versus controls in the lowest ADAMTS13 quintile was compared to those in the 2nd to 5th quintiles. Adjustment was performed for VWF antigen (VWF:Ag), factor VIII activity (FVIII:C), blood group, and age. RESULTS: Forty-six of 208 patients (22%) showed ADAMTS13 levels below the 10th percentile, compared with 5 of 125 controls (4%; p < 0.001). Odds ratios/95% confidence intervals were 7.30/2.73-19.50 for the lowest percentile and 2.44/1.15-5.16 in the quintile comparison after adjustment for VWF:Ag, FVIII:C, blood group, and age. Comparing the proportion of patients with ADAMTS13 activity below the 10th percentile within the different stroke subtypes (undetermined, cardioembolic, steno-occlusive arteriopathies), no statistically significant differences were found (undetermined, 16 of 89; cardioembolic, 6 of 40; steno-occlusive arteriopathies, 24 of 79; p = 0.08). ADAMTS13 levels did not significantly differ among stroke subtypes (p = 0.29). INTERPRETATION: Our findings implicate reduced ADAMTS13 activity as a risk factor for pediatric AIS, and support the concept that ADAMTS13 has a role in the pathogenesis of pediatric AIS. ANN NEUROL 2012.Annals of Neurology 08/2012; · 11.91 Impact Factor
Page 1
Molecular Biology of ADAMTS13
and Diagnostic Utility of ADAMTS13
Proteolytic Activity and Inhibitor Assays
Suresh G. Shelat, M.D., Ph.D.,1,3Jihui Ai, M.D., Ph.D.,1
and X. Long Zheng, M.D., Ph.D.1,2,3
ADAMTS13, a reprolysin-like metalloprotease, limits platelet-rich thrombus
formation in the small arteries by cleaving von Willebrand factor (vWF) at the Tyr1605-
Met1606 peptide bond. Deficiency of plasma ADAMTS13 activity, due to either an
inherited or an acquired etiology, may lead to a potentially lethal syndrome, thrombotic
thrombocytopenic purpura (TTP). Molecular cloning and characterization of the
ADAMTS13 gene have provided further insight into the structure-function relationships,
biosynthesis, and regulation of the ADAMTS13 protease, in addition to understanding the
pathogenesis of TTP and perhaps other thrombotic disorders. ADAMTS13 consists of a
short propeptide, a typical reprolysin-like metalloprotease domain, followed by a disinte-
grin-like domain, first thrombospondin type 1 (TSP1) repeat, Cys-rich domain, and spacer
domain. The carboxyl terminus of ADAMTS13 has seven more TSP1 repeats and two
CUB domains. ADAMTS13 is synthesized mainly in hepatic stellate cells, but also in
vascular endothelial cells. Recognition and cleavage of vWF require the proximal carboxyl
terminal domains, but not the middle and distal carboxyl terminal domains. Cleavage of
vWF appears to be modulated by shear force, binding to platelet or platelet glycoprotein-
1ba, heparin, inflammatory cytokine (interleukin-6), and chloride ion. At the site of
thrombus formation, the ADAMTS13 may be inactivated by thrombin, plasmin, and
factor Xa. Having a sensitive and specific assay for ADAMTS13 activity is not only critical
to understand the basic biology of ADAMTS13 protease, but also to facilitate a more
timely and accurate clinical diagnosis of TTP, and to initiate potentially life-saving plasma
exchange therapy. Although many assays have been developed and tested for clinical
applications, the fluorescent resonance energy transfer-vWF73 assay appears to be the
simplest and most promising assay to date.
KEYWORDS: Thrombotic thrombocytopenic purpura (TTP), von Willebrand factor,
microvascular thrombosis, ADAMTS13, metalloprotease, assays, clinical application
Objectives: On completion of the article, the reader should be able to (1) understand certain aspects of ADAMTS13 biology underlying
thrombotic thrombocytopenic purpura, and (2) be familiar with current assays and clinical utility of ADAMTS13 activity and inhibitors.
Thrombotic Thrombocytopenic Purpura—2005; Editor in Chief, Eberhard F. Mammen, M.D.; Guest Editors, Hau C. Kwaan, M.D., Ph.D.,
Charles L. Bennett, M.D., Ph.D. Seminars in Thrombosis and Hemostasis, volume 31, number 6, 2005. Address for correspondence and reprint
requests: X. Long Zheng, M.D., Ph.D., Assistant Professor, Department of Pathology and Laboratory Medicine, The Children's Hospital of
Philadelphia and The University of Pennsylvania, 34th St. and Civic Center Blvd, Abramson Research Center 816G, Philadelphia, PA 19104.
E-mail: email@example.com.
Professor;3The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. Copyright # 2005 by Thieme Medical Publishers,
Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. 0094-6176,p;2005,31,06,659,672,ftx,en;sth01115.
1Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia;
Page 2
Accreditation: Tufts University School of Medicine (TUSM) is accredited by the Accreditation Council for Continuing Medical Education
to provide continuing medical education for physicians.
Credit: TUSM designates this educational activity for a maximum of 1 category 1 credit toward the AMA Physicians Recognition Award.
Each physician should claim only those credits that he/she actually spent in the activity.
The 13th member of a disintegrin-like and
metalloprotease with thrombospondin type 1 motifs
(ADAMTS) family, ADAMTS13,1–7limits platelet
aggregation and microthrombi in small arteries by cleav-
ing vWF at the Tyr1605-Met1606 bond.8,9Deficiency
of ADAMTS13 protease due to either an inherited5or
acquired etiology10may result in formation of dissemi-
nated microvascular thromboses, exhibiting a potentially
lethal syndrome, thrombotic thrombocytopenic purpura
TTP had classically been diagnosed using a pen-
tad of criteria: thrombocytopenia, microangiopathic he-
molytic anemia (MAHA), fever, renal dysfunction, and
neurological abnormalities.11However, the diagnosis
has been made recently in patients with only thrombo-
cytopenia and MAHA.14–17On a histological level,
these symptoms are attributable to the presence of
platelet- and von Willebrand factor (vWF) -rich micro-
thrombi in the terminal arterioles and capillaries of distal
organs, leading to organ failure, coma, and death.18
Until the late 1960s, TTP was virtually fatal, with a
mortality rate of >90%,19but with advances in plasma
exchange, the survival rate from acute TTP approaches
80 to 90%.20,21
Several reviews have summarized the roles of
vWF and ADAMTS13 in TTP.13,22–30This article
specifically focuses on certain aspects of ADAMTS13
biology: the molecular cloning, structure-function rela-
tionship, biosynthesis, regulation of proteolytic activity,
ADAMTS13 mutations found in congenital TTP pa-
tients, and the diagnostic utility of assays for plasma
ADAMTS13 proteolytic activity and inhibitors.
In 1996, two groups attempted to isolate a protease
from normal human plasma that cleaves vWF, although
the attempts were not successful initially. However,
much was learned about the biochemical properties
of their partially purified products.8,9The protease
displays a greater proteolytic activity when vWF is
subject to high shear stress or denatured by urea or
guanidine in the presence of low ionic strength. Divalent
cations such as Ba2þ, Zn2þ, Ca2þor Co2þ, are necessary
to activate the protease.8,9These early studies led to the
development of several
for determining the protease activity in human plasma
or for tracking the activity in the fractions dur-
ing subsequent chromatographic purification of the
In 2001, the vWF-cleaving metalloprotease was
finally isolated and its partial amino-terminal sequence
was determined.1,2This led to the identification and
classification of the vWF-cleaving metalloprotease as a
new member of the ADAMTS family, designated
ADAMTS13.1–4By a completely different approach,
the identical ADAMTS13 gene was also cloned.5
The ADAMTS13 gene is located on chromosome
9q34, spanning 37 kb in length and containing 29
exons.4,5Several alternatively spliced variants were found
during the cloning and sequencing.3–5Analysis of the
translated product showed that ADAMTS13 belongs to
the metzincin superfamily of zinc metalloproteases,
which, thus far, contains 19 family members that share
the common structure of a hydrophobic signal sequence,
a propeptide, a metalloprotease domain, first thrombo-
spondin-1 (TSP1) repeat, a disintegrin-like domain, a
cysteine-rich domain, and a spacer domain.3–5The
carboxyl terminus of ADAMTS13 contains seven
more TSP1 repeats and two CUB domains, which are
unique to ADAMTS13 and were previously identified in
complement components c1r and c1s, urinary EGF, and
bone morphogenetic protein.31
Northern blotting detects a 4.7-kb mRNA in human
liver,3–53.5- to 4.7-kb mRNAs in livers of different
mouse strains,32,33and a 2.4-kb mRNA in human
placenta and skeletal muscle.4In situ hybridization and
immunohistochemical staining with a monoclonal anti-
body directed against the disintegrin domain of
ADAMTS13 have localized the ADAMTS13 mRNA
and protein, respectively, to hepatic stellate cells (the
interstitial cells of liver parenchyma that are elongated
and varied in size and shape).34,35ADAMTS13-
expressing cells are also positive for a-smooth muscle
actin, suggesting only activated hepatic stellate cells or
myofibroblasts produce ADAMTS13.34Cell fractiona-
tion showed that full-length ADAMTS13 is synthesized
and secreted from primary hepatic stellate cells from
mouse35and rat (M. Niiya and X. Zheng, unpublished
data, 2005). Other cell types such as hepatocytes, sinus-
oidal endothelial cells, or Kupffer cells do not express
detectable amounts of ADAMTS13 intracellularly or in
the culture medium.35Furthermore, cell lines derived
from normal hepatocytes (THLE-3) or hepatocellular
carcinoma (HuH-7) do not express ADAMTS13, but
those derived from hepatic stellate cells of human
(hTERT-HSC) and rat (CFSC-3H and -8B) do secrete
Page 3
active ADAMTS13 into the culture medium.35These
data further support that activated hepatic stellate cells
are the major ADAMTS13-producing cells in the liver.
Activated hepatic stellate cells have been implicated in
the development of liver fibrosis and cirrhosis36–38after
various insults because of their ability to produce and to
deposit extracellular matrix proteins such as fibril colla-
gen36–38in the injured area. Thus, upon activation, the
enhanced proteolytic activity of ADAMTS13 in the
hepatic stellate cells may be associated with either
formation or resolution of liver fibrosis. More studies
are needed to delineate the relationship between
ADAMTS13 protease and liver fibrosis.
In addition to hepatic stellate cells, platelets or
other tissues may also produce and secrete ADAMTS13.
Reverse transcriptase polymerase chain reaction (RT-
PCR) using a single-strand cDNA can detect the
ADAMTS13 mRNA transcripts in almost all major
organ tissues, including human heart, lungs, pancreas,
brain, kidneys, adrenal glands, placenta, uterus, ovaries,
and prostate.5–7
ADAMTS13 mRNA suggests that vascular endothelial
cells may produce ADAMTS13. Our results (D. Shang,
X.W. Zheng, and X. Zheng, unpublished data, 2005)
showed that primary human umbilical cord vein endo-
thelial cells, primary aortic endothelial cells, and micro-
vascular endothelial cell lines (EVC304) all synthesize
and secrete full-length ADAMTS13, that is, detected
by RT-PCR, Western blotting with rabbit anti-
ADAMTS13, and capable of proteolytic cleavage of
vWF and vWF73 substrates. Considering the enormous
surface area covered by endothelial cells, it is possible
that the plasma pool of ADAMTS13 may be derived
from the vascular endothelial cells, in addition to hepatic
stellate cells.
In a pulse-chase experiment, ADAMTS13 is
readily secreted and can be detected within 3 hours of
chase in the conditioned medium of transiently trans-
ADAMTS13 migrates at 190 kd on a sodium dodecyl
sulfate (SDS) polyacrylamide gel,39–41similar to that of
ADAMTS13 purified from plasma,1–3although the
calculated molecular weight is only 150 kd.3–5The
difference in molecular mass is attributed to glycosyla-
tion4,39,42and perhaps other posttranslational modifica-
tions.4Recombinant ADAMTS13 secreted into the
conditioned medium is resistant to endoH digestion,39
but sensitive to N-glycanase F and sialidase,39suggesting
the presence of a complex-type structure of N-linked and
O-linked oligosaccharides. ADAMTS13 is localized to
the endoplasmic reticulum and Golgi apparatus, consis-
tent with the distribution of secretory proteins.40
Despite structural homology to ADAMTS1, which
interacts with the extracellular matrix via its spacer
domainand thrombospondin
ADAMTS13 expressed from transfected COS7 and
distribution of
type repeats,43,44
RFL-6 cells does not bind detectably to either cell
membrane or extracellular matrix.40
The role of the propeptide in biosynthesis
and secretion of ADAMTS13 has been investigated
recently. The ADAMTS13 propeptide is unusually
short (?41 amino acid residues) compared with the
typical ?200 residues in other members of the
ADAMTS or ADAM protease family.45–47
domain terminates in a typical propeptide cleavage
sequence, RQRR74. The furin
required for cleavage of ADAMTS13 propeptide,39
since changing this sequence to KQDR results in secre-
tion of pro-ADAMTS13 with intact propeptide
attached.39For several related proteases, propeptide is
required for folding and maintaining enzymatic latency
during biosynthesis. However, deletion of the propep-
tide did not impair the secretion of active ADAMTS13
in transfectedHeLa cells,
ADAMTS13 propeptide is not required to maintain
enzyme latency.39
consensus sequence is
The mature form of the ADAMTS13 protease starts
with the metalloproteases domain that has three His
residues (HEXXHXXGXXHD) that coordinate the
essential Zn2þ
or Ca2þbinding.4
Met249 that forms a Met-turn and the residues forming
a predicted Ca2þcoordination site (Glu83, Asp173,
Cys281, Asp284) are also present in the metalloprotease
domain.4The binding sites for Ca2þand Zn2þare
consistent with data showing that the enzyme is inhib-
ited by their chelation.40,41,48At the concentration of
16 nM (three to four times higher than plasma concen-
tration), metalloprotease domain alone does not cleave
multimeric vWF or small peptidyl substrate GST-
vWF73-H that contains 73 amino acid residues from
the central A2 domain of vWF, flanked by a glutathione
s-transferase (GST) at N-terminus and 6xHis epitope
at C-terminus (Fig. 1).49However, with a prolonged
incubation (16 to 24 hours), the metalloprotease domain
does cleave GST-vWF73-H substrate at a site other
than the Tyr-Met bond, generating a N-terminal
fragment ?3 kd smaller than expected,49suggesting
the carboxyl-terminal domains are required for substrate
recognition and specificity.
Remarkably, addition of one or several proximal
carboxyl terminal domains (including the disintegrin
domain, first TSP1 repeat, the Cys-rich domain, and
spacer domain) to the metalloprotease domain of
ADAMTS13 restores the specific cleavage of Tyr-Met
bond of GST-vWF73-H substrate.49However, the
proteolytic activity of the ADAMTS13 mutants toward
both vWF and GST-vWF73-H (Fig. 1) or fluorescent
resonance energy transfer (FRETS)-vWF73 is not
fully restored until all the proximal carboxyl-terminal
The conserved
Page 4
domains are added.49These data suggest that all of the
proximal carboxyl-terminal domains participate in sub-
strate recognition and are required for cleavage of vWF.
Further addition of the TSP1 2 to 8 domains and two
CUB domains does not increase proteolytic cleavage of
vWF,40,41,49and GST-vWF73 or FRETS-vWF73.49
Several strains of mice (BALB/c, C3H/He, C57BL/6,
and DBA/2) lack the carboxyl-terminal domains from
the seventh TSP1 repeat to the CUB domain.33These
data support the notion that the middle and the distal
carboxyl-terminal domains may be dispensable at least in
vitro in humans and in vivo in mice.
Full-length ADAMTS13 can bind immobilized vWF
with a stoichiometry of 1:2 (i.e., one ADAMTS13
molecule binds two vWF molecules), reaching an equili-
brium in ?2 hours, with a half-life of dissociation
of ?4 hours.39,49The estimated Kdfor ADAMTS13
binding to vWF is ?14 nM, comparable to Km of
16 nM, determined independently.39ADAMTS13 trun-
cated after the spacer domain binds vWF comparably to
Removal of the spacer domain significantly impairs its
binding to vWF,39suggesting that the spacer domain
contributes substantially to ADAMTS13-vWF interac-
tion. The metalloprotease domain at the highest concen-
The binding interaction between ADAMTS13
and GST-vWF73-H has been determined recently.
vWF73 (D1659-R1668) has been shown to be the
minimal and essential peptide region of vWF being
recognized and cleaved by ADAMTS13.50Full-length
Figure 1
construct of full-length (FL) ADAMTS13 and carboxyl-terminal truncated ADAMTS13 variants. (B) Cleavage of FL vWF and GST-vWF73-
H by ADAMTS13 and C-terminal truncated variants. delCUB, aal-1191; MDTCS, aal-685; MDTC, aal-554; MDT, aal-439; MD, aal-385; M,
metalloprotease. (Adapted from Zheng X, Nishio K, Majerus EM, Sadler JE. Cleavage of von Willebrand factor requires the spacer
domainof the metalloprotease ADAMTS13. J Biol Chem 2003;278:30136–30141 and Ai J, Smith P, WangS, et al. The proximalcarboxyl
terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor. J Biol Chem
Cleavage of von Willebrand factor (vWF) and vWF73 by ADAMTS13 and variants. (A) Schematic representation of the
Page 5
ADAMTS13 and ADAMTS13 truncated after the
spacer domain bound GST-vWF73-H with Kd (s)
of ?4.6 and ?7.0 nM, respectively.49The affinity of
binding ADAMTS13 or the carboxyl-terminal trun-
cated mutant to vWF73 is approximately three times
higher than that to vWF,39,49suggesting that the do-
mains surrounding the central A2 subunit of vWF
negatively regulate the interaction of ADAMTS13
with vWF.
The relative contribution of each of the proximal
carboxyl-terminal domains in substrate binding has also
ADAMTS13 expressed from Escherichia coli and re-
folded in vitro. The binding of ADAMTS13 domains
to GST-vWF73-H was performed on an enzyme-linked
immunosorbent assay (ELISA) plate and in solution.
The Kdvalues for the disintegrin domain (D), the first
TSP1 repeat (T), the Cys-rich domain (C), and the
spacer domain (S) or DTCS fragment to bind GST-
vWF73-H are ?489, ?136, ?121, and ?108 or 13 nM,
respectively (Table 1).49The metalloprotease domain
does not bind detectably at the concentrations tested.
These data suggest that each of the proximal carboxyl-
terminal domains participates in substrate recognition
and acts cooperatively to confer efficient binding, con-
sistent with the proteolysis of vWF and vWF73 by
various ADAMTS13 mutants described above.49
recombinant domains of
Cleavage of vWF by ADAMTS13 may be modulated by
flow shear stress,9,10,51structural elements of vWF,48
heparin sulfate, platelets or platelet glycoprotein-1ba
(GP1ba),48sodium chloride,52and inflammatory cyto-
kines.53vWF is not cleaved under physiological con-
ditions due to structural inaccessibility of the binding or
cleavage site on the vWF molecule by ADAMTS13.
Modeling of the vWF-A2 domain suggests that the
Tyr1605-Met1606 bond lies buried within the core b
sheet of the native structure.54Shear force and denatur-
ing reagents, or binding of platelets that expose
the ADAMTS13 binding and cleavage site on vWF,
significantly enhance the
ADAMTS13.8–10However, even in the presence of
denaturing reagents, the ADAMTS13 binding site
appears not fully exposed. For example, the rate of
cleavage of the A2A3 mutant is ?10-fold higher than
that of the A1A2A3 mutant,48suggesting that the A1
domain may block the access of ADAMTS13 to
the cleavage site. This blockage can be removed by
interaction with platelet GP1ba or unfractionated
Other factors in the milieu may regulate
ADAMTS13-vWF interaction, such as chloride ions52
and hemoglobin.55The cleavage of vWF in the absence
of NaCl at pH 8.0 occurs with apparent kcat/km?3.4?
104M?1s?1, but this value decreases ?10-fold in the
presence of 0.15 M NaCl.52The inhibitory effect can be
found with a relative potency of ClO–4>Cl–>F–.52
Inhibition of ADAMTS13 activity has been observed
by hemoglobin at concentrations exceeding 2 g/L,
which occurred during severe hemolysis in a fatal
case of congenital TTP.55Unlike many other members
of ADAMTS family that form SDS-stable complexes
with a2-macroglobulin, a nonspecific protease inhi-
bitor present in the serum of healthy individuals,
ADAMTS13 does not bind a2-macroglobulin detect-
ably.40Inflammatory cytokines such interleukin-6, but
not interleukin-8 and tumor necrosis factor-a, inhibit
the cleavage of ultralarge vWF (ULvWF) under flow.53
The findings suggest a potential linkage between in-
flammation and thrombosis that may be of therapeutic
Thrombin, factor Xa (FXa) and plasmin may
regulate ADAMTS13 activity at the site of thrombus
formation. ADAMTS13 is proteolytically cleaved
following activation of coagulation in human plasma.56
Incubation of purified recombinant ADAMTS13 with
various concentrations of human thrombin (0.9 to
90 nM) results in degradation of ADAMTS13 in a
time- and concentration-dependent fashion.56Such
cleavage can be abrogated when thrombin binds
soluble thrombomodulin, but not heparin, suggesting
the involvement of thrombin exosite I, but not exosite
II, in ADAMTS13 recognition. Plasmin and FXa
also cleave ADAMTS13 and give rise to a similar
but not identical fragmentation pattern, resulting
in loss of ADAMTS13 activity.56Plasmin cleaves
cleavage of vWFby
Table 1
vWF and GST-vWF73-H
Binding of ADAMT13 and Various Mutants to
CysR ND
Spa ND
Binding of FL ADAMTS13 and various ADAMTS13 mutants to the
purified plasma vWF and GST-vWF73-H was determined by ELISA
method and the values for Kd(s) were determined by fitting into a
double-reciprocal version of the binding equation. MDTCS and DTCS
indicate the ADAMTS13 mutant truncated after the spacer domain
with and without the metalloprotease domain, respectively. M, D,
TSP1, CysR, S were all tagged at the N-termini (except for M) by
V5-His epitope derived from vector. M was tagged at the C-terminus
by V5-His.
vWF, von Willebrand factor; FL, full-length; GST, glutathione
s-transferase; MDTCS, XXX; ND, not determined; DTCS, XXX; M,
metalloprotease; Dis, disintegrin domain; TSP1, thrombospondin
type 1; CysR, Cys-rich domain; Spa, spacer domain; ELISA,
enzyme-linked immunosorbent assay.
Page 6
ADAMTS13 faster than do thrombin and FXa,56
suggesting a role of ADAMTS13 and vWF in tissue
To date, more than 70 mutations on ADAMTS13 gene
have been described in patients with congenital or
familial TTP (Table 2).5,42,57–61The majority of these
mutations are missense mutations (?50%), followed
by splice site mutations, silent mutations, nonsense,
and frameshifts.5,13,42The mutations are distributed
throughout the ADAMTS13 gene with no apparent
clustering or hot spots. Twelve missense mutations
involving cysteine residues have been identified in
patients with familial TTP (Table 2), suggesting the
importance of proper formation of disulfide bridges in
ADAMTS13 function.
Half of the patients with familial TTP experience
their acute episodes during childhood and half during
adulthood.62In the latter patients, these episodes may be
accompanied by pregnancy, infection, or other condi-
tions that may result in the production of inciting
factors.62Although most of these mutations have been
identified in patients with familial TTP, it is unclear if
they also contribute to acquired TTP, which represents
the majority of TTP cases.
Several missense mutations have been character-
ized by site-directed mutagenesis of the ADAMTS13
gene, followed by transfection into different expression
systems, and assaying for their biosynthesis, secretion,
and activity in the conditioned medium. Several re-
R268P, and C508Y42exhibit low or no secretion and
little activity in the conditioned medium of transfected
cells. The R193W59and the A250V63mutations ex-
hibited decreased secretion as compared with the wild
type ADAMTS13. The R193W59is close to the active
site of the metalloprotease domain, whereas the A250V
is near the Met249necessary for Zn2þbinding, suggest-
ing that the small amount of protease secreted may also
have compromised proteolytic activity. Similarly, the
R268P substitution impairs secretion of the mutant
protein from cells42,59perhaps due to the disruption of
the a-helix structure, which may account for its dimin-
ished activity in plasma. On the other hand, the re-
combinant P475S is secreted normally in transfected
cells, but shows low proteolytic activity toward vWF.42
Interestingly, approximately 9.6% of Japanese may be
heterozygous for this polymorphism, which may be
responsible for reduced protease activity.42However,
despite of a high prevalence of P475S in the Japanese
population, such a mutation with low proteolytic activity
is rare in whites (0.5%)64and in Chinese (1.7%).65
Similarly, the nonsense mutation Q449X is also secreted
normally in transfected cells, but has very low activity,42
consistent with reports that the spacer domain is neces-
sary for ADAMTS13 activity.40,41,49,66
Another recombinant missense, Q448E, which is
secreted normally, has been reported as a single nucleo-
tide polymorphism (SNP),5,42although it retains full
activity in transfected cells.42Although numerous SNPs
have been reported (Table 2), none have yet shown a
clear relationship to TTP,28although it is possible they
may be associated with other conditions, such as coro-
nary artery disease, in which ADAMTS13 activity is
Several frameshift mutations have been identified,
and these aberrantly carboxyl-terminal truncated mu-
tants may demonstrate either impaired secretion and/or
low proteolytic activity. The 4143 to 4144insA mutation
that deletes the second CUB domain exhibits impaired
secretion, but retains proteolytic activity if the same
amount of protein is tested.60Several ADAMTS13
gene mutations have been identified at splice sites, which
disrupt the conventional GT-AG consensus splice se-
quences, and prevent production of normally spliced
Such alternatively spliced forms of
ADAMTS13 are present in normal human plasma,2
although the biological functions of these variants are
not known.
The in vitro expression studies indicate that
ADAMTS13 mutations exhibit a heterogeneous phe-
notype, depending on the effects on biosynthesis, secre-
tion, and protease bioactivity. These reflect the clinical
heterogeneity seen among hereditary TTP patients,
most of whom harbor compound heterozygous muta-
tions. The parents of hereditary TTP patients (asymp-
tomatic carriers) have about a 50% level of ADAMTS13
activity, although some asymptomatic carriers exhibit
very low levels of ADAMTS13 activity (less than 10% of
normal) who are compound heterozygotes such as
R268P/P475S.69Of note, most of the described homo-
zygous mutations,such
414þ1G>A splice,68L232Q,70and 1783delTT,71
have some evidence of a consanguineous pedigree, and
there are very few null alleles, suggesting that a complete
lack of ADAMTS13 protease may be lethal.5,13,28,42,59
as R692C,5
ADAMTS13 activity in normal plasma is reported to be
50 to 178%.14Severe deficiency of plasma ADAMTS13
activity toward vWF has been considered to be the
primary cause of congenital and acquired idiopathic
TTP,5,10,11,72,73although mildly to moderately reduced
levels of ADAMTS13 have been observed in conditions
such as inflammation, coronary artery disease,67preg-
nancy, liverdisease,74disseminated intravascular coagula-
tion,75sepsis, and heparin-induced thrombocytopenia.76
The clinical utility of the ADAMTS13 proteolytic
Page 7
Table 2Location and Functional Consequence of ADAMTS13 Gene Mutation
Effect on
Amino AcidDomain
Activity in Human
Plasma (unless
''in vitro'' is noted) SecretionComment
50border In3 G>A63
2Q44X proP<3%80
In 3
In 4
Splice abolishedM<3%
Silent MSNP5,42
?25% of wildtype68
In 6
Del GþC
<3%42; <5%100;
in vitro: no activity42
R268P MNo42,68
In 8
Splice Dis
P353L Dis
In 9
W390X Tsp1–1
R398H Tsp1–12–7%
In 10
Splice abolishedTsp1–1 <3%
In 10
Splice Tsp1–1
Q448E Cys<3%5,69; in vitro,
full activity with
" lower molecular
weight vWF42
<3%69; in vitro:
very low activity42
Q449X Cys yes42
P457L Cys
P475SCys <3%5,42,69; in vitro:
very low activity,
with " lower
MW vWF subunits42
<3%42; In vitro:
no activity42
1523G>AC508Y CysNo42,68
1584þ5G>A5, or
þ106C>G5, or
þ 236T>C5
R528G Cys
In 13 Splice Cys2–7%
Page 8
activity assays remains the subject of considerable debate
in recent years,77–79partly due to the variability of each
assay. The assays can be categorized into direct versus
indirect and static versus flow based, depending on the
reaction conditions and how the cleavage of the substrate
is determined.
Direct Assays
Direct assays focus on detection of cleavage products
of a given substrate, whether it is a macromolecular
vWF, a vWF A2 domain, or the small peptide GST-
vWF-73 or FRETS-73. Agarose or polyacrylamide
gel electrophoresis (PAGE), Western blotting, and
FRETS techniques have been used to detect the cleav-
age products of the substrate. The FRETS-vWF73
detects proteolytic cleavage of the specific Tyr-Met
peptide bond by ADAMTS13 in real time. By far,
this assay may be the most sensitive and promising
of all.
This assay was initially developed by Furlan et al8to
measure ADAMTS13 activity in plasma. After purified
vWF is incubated with citrated plasma samples for
Table 2 (continued)
Exon/Intron Nucleotide
Effect on
Amino Acid Domain
Activity in Human
Plasma (unless
''in vitro'' is noted)SecretionComment
1783–1784 TT del71
SilentCys SNP5,42
15Frameshift Sp<0.1 U/mL
In 15
A596V Sp<5%
P618A SpSNP57
16R625H Sp
2376del26 (2376–2401D)5
A732V Tsp1–2SNP5,57
19 C758RTsp1–3<5%
Frameshift Tsp1–3
Silent Tsp1–3SNP5,57
19 Frameshift Tsp1–32–7%
A900V Tsp1–5SNP5
21C908Y Tsp1–5<3%No68
C951GTsp1–5 2–7%
In 22
C1024G Tsp1–72–7%
A1033TTsp1–7 SNP5
24 R1034XTsp1–7<2%
Silent Tsp1–7
25 Del 3252–3253CT
R1096XTsp1–8 <5%100
FrameshiftCUB1 2–7%
In 28
Frameshift CUB2<10%; in vitro activity
equals wildtype60
14% of wildtype60
Silent CUB2SNP5,42
*The missense (change in amino acid residue), nonsense (introduction of premature stop), frameshift/deletion, alternatively splicing and silent
(no amino acid change) mutations are indicated in column 3.
SNP, single nucleotide polymorphism; SP, signal peptide; ProP, propeptide; M, metalloprotease, Dis, disintegrin; Tsp, thrombospondin; Cys,
cysteine-rich domain; Sp, spacer. The superscript numbers in columns 2 through 7 are reference citations.
Page 9
approximately 24 hours, agarose gel electrophoresis
and Westernblottingwith
anti-vWF antibody are used to determine the pro-
teolytic degradation of purified vWF multimers. The
relative amount of ADAMTS13 activity is calculated
from the reference of serially diluted normal human
This assay laid a strong foundation for under-
standing the pathogenesis of congenital or familial
TTP72,80–82and acquired TTP.80It has also provided
a tool to monitor the proteolytic activity of each fraction
duringthe subsequent
ADAMTS13 protease from normal human plasma.2
However, this assay is very complex and requires tech-
nical expertise in handling agarose gel electrophoresis,
transfer and Western blotting.
successfulpurification of
Tsai et al9,10,83initially developed this assay based on
detection of dimeric vWF fragments of 176 and 140 kd
on denatured, but unreduced SDS-PAGE and Western
blotting. This assay differs from the SDS-agarose
method developed above by Furlan et al8,72,80in these
ways: (1) no barium chloride activation is required;
(2) the incubation is relatively short (1 to 3 hours at
37?C); (3) it is easier to handle gels from SDS-PAGE
than agarose-gel electrophoresis; and (4) there is direct
visualization of the cleavage product, eliminating the
possibility of nonspecific degradation of the multimers.
This SDS-PAGE assay detected severe deficiency of
ADAMTS13 activity (<5% of normal) in all 39 samples
from 37 patients with idiopathic TTP.10In addition, the
inhibitory activity against ADAMTS13 protease was
detected in 26 of 39 samples (67%).10This assay is
sensitive enough to differentiate patients with congenital
TTP from asymptomatic carriers and healthy individu-
als, and allows for the precise mapping of the defective
ADAMTS13 gene.5
Alternatively, the epitope-tagged vWF fragment
(His-A2 domain or GST-vWF73) could be digested by
plasma ADAMTS13 within 1 to 3 hours.49,50,84The
cleavage of the Tyr-Met bond is determined by Western
blotting with an anti-His84or anti-GST49,50antibody.
The assay using the vWF A2 domain detected 13 of the
16 samples (81%) with an activity of <6%.84The GST-
vWF73 substrate is not cleaved by plasma from patients
with congenital or acquired TTP, but is cleaved by
plasma from patients with hemolytic uremic syndrome,
suggesting that GST-vWF73 is a specific substrate for
ADAMTS13. However, truncated ADAMTS13 mu-
tants after the Cys-rich domain may cleave GST-
vWR73.49Therefore, normal proteolytic cleavage does
not rule out the possibility of the functional abnormality
of ADAMTS13 protein due to mutations or acquired
autoantibodies that are directed against the middle and
distal carboxyl terminus. Again, the cleavage of a small
recombinant domain or a peptidyl substrate does not
require denaturing reagents and is free of contaminating
ADAMTS13 in the substrate.
FRETS-vWF73 is a chemically modified vWF73 de-
rived from the central A2 domain of vWF (D1596-
R1668) containing A2pr (Nma) and A2Pr (Dnp; Fig. 2).
It is readily cleaved by normal human plasma, but not by
ADAMTS13-deficient plasma.85It could differentiate
congenital and acquired TTP from patients with
hemolytic uremic syndrome (HUS) and heterozygous
mutations of ADAMTS13 gene or healthy individuals.85
The proteolytic cleavage of the FRETS-vWF73 is
acid.85Compared with the collagen-binding assay (de-
scribed below) and the GST-vWF73 assay (described
above), FRETS-vWF73 is superior. It is sensitive and
specific, requiring only 2 to 5 mL of citrated plasma for
each activity assay, and the cleavage of the substrate can
Figure 2
amino acid residues Q1599 and N1610 within the vWF peptide (D1595-R1668) are replaced by A2pr (Nma) and A2pr (Dnp), respectively.
When the Nma group is excited at 340 nm, fluorescence resonance energy is transferred to the neighboring quencher, Dnp. If the Tyr-
Met peptide bond (indicated by arrow) is cleaved by ADAMTS13, the energy transfer quenching the fluorescence does not occur,
allowing the emission of fluorescence at 440 nm from Nma (Adapted from Kokame K, Nobe Y, Kokubo Y, et al. FRETS-VWF73, a first
fluorogenic substrate for ADAMTS13 assay. Br J Haematol 2005;129:93–100.)
Schematic representation of fluorescent resonance energy transfer (FRETS)-von Willebrand factor (vWF) 73 substrate. The
Page 10
be monitored in real time with aresult in ?1 hour.85The
FRETS-vWF73 can identify more TTP patients with
severe ADAMTS13 deficiency and positive inhibitor,
which can be verified independently by Technozyme-
ELISA (Baxter, Orth, Austria) and immunoprecipita-
tion and Western blotting methods (S. Shelat, P. Smith,
J. Ai, and X. Zheng, unpublished data, 2005).
Indirect Assays
Indirect assays depend on measuring the residual sub-
strate or disappearance of the substrates. This type
of assay may be influenced by non-specific proteolysis
of vWF by other enzymes in plasma, unless they
are inactivated by incubation of plasma with Pefo-
bloc73,80,81,86or other serine protease inhibitors.17,87
Initially described by Gerritsen et al88and modified by
many others,17,74,87,89this assay is based on preferential
binding of large vWF multimers to type III human
collagen. Afterincubation
ADAMTS13-depleted plasma with patient's plasma or
normal human plasma (as control) in the presence of
5 mM Tris-HCl (pH 8.0), 1.5 M urea, and 10 mM
BaCl2 for 16 to 24 hours, the amount of residual
vWF is determined by binding to human collagen
type III. Bound vWF is quantified by peroxidase-
conjugated anti-vWF immunoglobulin G (IgG) on an
ELISA plate, followed by a densitometry measure-
This assay is relatively straightforward to perform
and can accommodate many samples simultaneously. It
is quite robust in the detection of severe deficiency of
ADAMTS13 (16 of 20 patients) and high titer inhibitor
(seven of 16 patients) in idiopathic TTP.17It correlates
with other indirect assays well.91The presence of in-
hibitor detected by this assay correlates with more
relapses,17,87whereas patients with severe ADAMTS13
deficiency but no detectable inhibitor have a better
response and outcome,17suggesting that the assay may
be used for making a diagnosis, monitoring therapy, and
predicting outcome.
ofpurifiedvWF or
After digestion of the purified vWF in the buffer (similar
to that in collagen-binding assay), the residual vWF is
determined by a ristocetin cofactor activity in a platelet
aggregometer. The method is reproducible as shown by
low intra- and interassay coefficients of variation (2.8%
and 7.5% for normal samples, respectively, and 8.7% and
12.9% for abnormal samples, respectively).92Further-
more, it detects severe ADAMTS13 deficiency in pa-
tients with acute, classic TTP. The majority of patients
with low titer inhibitor respond to plasma exchange
treatment with increases in ADAMTS13 activity,
whereas ADAMTS13 deficiency persists in some pa-
tients with high titer inhibitor despite clinical remis-
sion.92This assay has not been widely used because only
limited samples can be analyzed at one time on the
Two types of assays use flow conditions for measuring
ADAMTS13 activity. First, Shenkman et al93used a
cone and plate(let) analyzer to evaluate the ability of
TTP plasma to increase deposition on a polystyrene
surface from normal plasma under flow conditions.
This assay is sensitive and specific for detection of
ADAMTS13 deficiency and inhibitors.93Increased de-
position of normal platelets was observed following the
addition of plasma in all 15 acute TTP patients, but only
in three of 14 patients in remission.93The IgG from
plasma or serum of three of five acute TTP patients
enhanced platelet deposition,93suggesting the possibility
of detecting autoantibodies against ADAMTS13 by this
method. Second, Dong et al94described that ULvWF
multimers, upon release from cultured endothelial cells,
form extremely long, platelet-decorated string-like
structures on the surface of endothelial cells under fluid
shear stress. When perfused with normal human plasma,
the string-like structures disappear rapidly within a few
minutes, indicating the cleavage of ULvWF by
ADAMTS13. Under flow conditions, cleaving ULvWF
occurs at 1000-fold faster kinetics than that observed
under static conditions. However, plasma from patients
with idiopathic TTP does not cleave these platelet-
decorated strings.
This assay uses epitope-tagged recombinant fragment
(vWF A2 domain or vWF73) as a substrate.84,95The
substrate is immobilized to a microtiter plate with an
antibody to one tag. After incubation of the immobilized
substrate with plasma samples, an antibody to another
tag detects the residual substrate. The proteolytic activity
of ADAMTS13 is inversely proportional to the residual
substrate concentration. This type of assay is relatively
simple and sensitive. For example, the ADAMTS13
activity in patients with TTP detected by vWF
A2 domain-ELISA is 2.4?0.7%, compared with
40?4.2% in normal human plasma.84The vWF73-
ELISA can differentiate TTP patients from carriers
of ADAMTS13gene mutation and normal healthy
The ADAMTS13 Inhibitor Assay
ADAMTS13 autoantibodies may be classified into two
groups: inhibitory antibodies or noninhibitory antibod-
ies, depending on whether the autoantibodies block
proteolyticcleavage of vWF(or other peptidyl
Page 11
substrate) in an in vitro assay. Antibodies that react
proximally to ADAMTS13 may block proteolytic
activity in vitro, whereas antibodies that target to the
middle and distal carboxyl terminus of ADAMTS13
may not, based on the structure-function analysis of
ADAMTS13.40,41Thus far, all inhibitory antibodies
react with the Cys-rich and spacer domains with
some specificity toward the catalytic domain and
other parts of the molecule,96supporting the critical
function of the Cys-rich and spacer domains in substrate
All of the functional assays for ADAMTS13
activity are capable of detecting the inhibitory autoanti-
bodies with a wide range of sensitivities. The FRETS-
vWF73 appears, by far, the most sensitive test among
three assays we evaluated (collagen-binding assay,
GST-vWF73, and FRETS-vWF73) to identify the
inhibitory autoantibodies (S. Shelat, P. Smith, J. Ai,
and X. Zheng, unpublished observation, 2005). An
ELISA assay along with Western blotting using the
recombinant ADAMTS13 as an antigen has been de-
veloped recently, which can detect both inhibitory and
noninhibitory autoantibodies.97
found in 97% of untreated patients with acute acquired
TTP whose plasma levels of ADAMTS13 are below
10%.97The corresponding prevalence of IgM antibodies
is 11%.
The presence of ADAMTS13 autoantibodies are
rather specific for making a diagnosis of acquired
TTP.10,78In addition, a high titer of inhibitory autoanti-
bodies correlates with more relapses. Adjunct immune
therapies such as rituximab, an anti-CD20 chimeric
monoclonal antibody, or cyclophosphamide, may be
considered once inhibitory autoantibodies are identified
in patients with acute TTP who do not adequately
respond to plasma exchange or are chronically re-
lapsed.17,87,98,99Therefore, a robust ADAMTS13 in-
hibitor assay is critical for understanding of the
mechanism of TTP and for tailoring therapy.
In conclusion, molecular cloning and character-
ization of ADAMTS13 gene and protein structure have
opened a new avenue for study of the biology and
biochemistry of the ADAMTS13 protease. Develop-
ment of a sensitive and specific assay for ADAMTS13
activity and inhibitor would not only help to understand
the pathogenesis of TTP and other thrombotic disor-
ders, but also to facilitate a more timely and accurate
clinical diagnosis, which is crucial for initiating and
tailoring therapy in patients with TTP.
IgG antibodies are
This work is supported in part by grants from National
Institute of Health (HL 079027 to XZ and HL 075246
to SS), American Heart Association Begin grant-in Aid,
and National Blood Foundation.
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enzyme immunoassayof


text:
Series starts 11/28
Game 1: Sunday, November 28th | ||
Knicks | 125 | Final 2OT |
Pistons | 116 | |
Recap »Boxscore » |
Game 2: Sunday, January 30th | ||
Pistons | 106 | Final |
Knicks | 124 | |
Recap »Boxscore » |
Game 3: Friday, March 18th | ||
Knicks | 95 | Final |
Pistons | 99 | |
Recap »Boxscore » |
Coverage: MSG
7:30 PM ET, March 18, 2011
The Palace of Auburn Hills, Detroit, MI
AUBURN HILLS, Mich. -- Detroit's newest starting lineup worked about as well as the previous 18, so coach John Kuester turned to his backups for a fourth-quarter rally.
Charlie Villanueva, Chris Wilcox and Will Bynum were up to the challenge.
Villanueva scored 11 of his 14 points in the fourth quarter, and Wilcox threw down a one-handed dunk off a missed shot with 53 seconds left, helping the Pistons come back from an 11-point deficit for a 99-95 win over the New York Knicks on Friday night.
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•Knicks blog | ESPN New York
Bynum had five points and five assists in the fourth -- after sitting out the first three periods.
"We've been communicating with our guys, telling them all the time that you have to be ready," Kuester said. "Will hadn't played ... and he came in and we need him and he was phenomenal."
Carmelo Anthony scored only six points for New York and missed all five of his shots in the fourth. After Wilcox's dunk gave Detroit a two-point lead, Anthony drove past Tayshaun Prince, but Wilcox was there defensively to force a missed shot.
After Bynum missed on the Pistons' next possession, Wilcox helped keep the ball alive, and Detroit came up with it. Bynum made two free throws with 11 seconds left for the final margin.
The Knicks have lost four of five and appear likely to finish sixth or seventh in the Eastern Conference. The loss to the lowly Pistons won't help.
"They're a pretty good ball team, but they're not in the playoff hunt. We are," New York's Amare Stoudemire said. "We're trying to get better seeding, so to lose tonight is definitely not a proud moment for us."
Anthony finished 2-of-12 from the field and walked off toward the team bus after the game while reporters spoke with teammate Chauncey Billups.
"He just really didn't get a lot of opportunities at the basket," Billups said. "They denied him, they fronted him when he tried to take them down to his sweet spot, they were bringing a guy over playing behind him and he was just getting a lot of attention."
Anthony tried to post up the smaller Rodney Stuckey during the fourth quarter, to no avail.
Detroit's Richard Hamilton missed the game because of the death of his grandfather, and the Pistons started an odd lineup in his absence. Detroit's backcourt of 6-foot-8 Tracy McGrady and 6-11 Austin Daye was the tallest to start a game in the NBA since at least 1986-87, according to STATS, LLC.
It was the 19th different starting lineup this season for the Pistons, who have had trouble finding many combinations that work.
Trailing 78-70 after three quarters, Kuester started the fourth with five reserves in the game. He made only one substitution the entire period, putting Prince -- a starter -- back in for the final 1:40.
Detroit trailed 81-70 early in the fourth before going on a 19-5 run. Villanueva made three 3-pointers during that stretch, and Bynum capped the run with a reverse layup and then a free throw moments later to put the Pistons ahead 89-86.
The Knicks moved back ahead, but Detroit tied it when Billups missed an outside shot, and Stuckey beat the Knicks down the court for a layup to make it 95-all.
Wilcox finished with 13 points and 12 rebounds, one of six Pistons in double figures. Stoudemire and Toney Douglas scored 20 points apiece for New York.
Billups scored 12 points against his former team but turned the ball over eight times.
Detroit scored eight straight points early in the third quarter to take a 63-55 lead, but after Stoudemire and Prince traded baskets, the Knicks went on a 17-0 run. Billups scored the first seven points, then Stoudemire put New York ahead with a powerful one-handed dunk over Greg Monroe while drawing a foul. The three-point play gave the Knicks a 67-65 lead.
Douglas made a 3-pointer from the right corner, and Shawne Williams added a dunk on a fast break to push the lead to seven. Douglas scored again before Detroit finally scored with 1:30 left in the period when New York was called for a defensive 3-second violation and Ben Gordon made the free throw.
Game notes
The Knicks shot 12-of-31 from 3-point range, a day after making a team-record 20 shots from beyond the arc in a win over Memphis. Douglas made nine 3-pointers in that game. He made four against the Pistons. ... Prince led Detroit with 16 points.
Team Stat Comparison
Points | 95 | 99 |
Rebounds (Offensive-Total) | 13-42 | 12-38 |
Assists | 23 | 26 |
Turnovers | 18 | 13 |
Steals | 6 | 14 |
Blocks | 4 | 5 |
Fast Break Points | 7 | 19 |
Fouls (Tech/Flagrant) | 17 (1/0) | 17 (0/0) |
Largest Lead | 11 | 8 |
Top Performers
Game Leaders
Points | A. Stoudemire 20 | T. Prince 16 |
Rebounds | A. Stoudemire 12 | C. Wilcox 12 |
Assists | T. Douglas 11 | W. Bynum 5 |
Steals | J. Jeffries 2 | T. McGrady 4 |
Blocks | J. Jeffries 1 | B. Wallace 2 |
Team rosters: New York | Detroit |
Next 5 Games
Research Notes
Carmelo Anthony had just 6 points in 37 minutes vs the Pistons. That is his fewest points scored in his career with at least 30 minutes played. |
ESPN Stats & Information |


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