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Heparin Cofactor II Inhibits Arterial
Thrombosis After Endothelial Injury
Doug Tollefsen
Hematology Division
Washington University
St. Louis, MO
Heparin Cofactor II
• 66-kDa glycoprotein
Synthesized by the liver
Plasma concentration ~1 µM
Half-life ~2.5 days
Extravascular distribution ~40%
• Member of the serpin family
Specifically inhibits thrombin (not Xa, IXa, etc.)
Rate of inhibition increased ~10,000-fold by
dermatan sulfate or heparin
Covalent thrombin-HCII complexes cleared
rapidly by hepatic LDL receptor-related
protein (LRP)
Inhibition of Thrombin Added to Plasma
Containing Heparin
kDa
96 125I-thrombin-HCII
85 125I-thrombin-AT
32 125I-thrombin
(B chain)
.0001 .001 .01 .1 1 2 5 10 20 50
Heparin (units/ml)
Inhibition of Thrombin Added to Plasma
Containing Dermatan Sulfate
kDa
96 125I-thrombin-HCII
85 125I-thrombin-AT
32 125I-thrombin
(B chain)
.01 .1 1 10 100 880
Dermatan Sulfate (µg/ml)
Structures of Heparin Cofactor II
and Antithrombin
GAG- binding Reactive
Domain Site
1 114 136 393 432
P1 Arg
AT
Acidic GAG- binding Reactive
Domain Domain Site
1 53 75 173 193 444 480
P1 Leu
1 2 HCII
“hirudin-like”
N
1
2 Exo I Allosteric mechanism
HO- S • Dermatan sulfate binding enables
L
“hirudin-like” domain 1 of HCII to
C Exo II interact with exosite I of thrombin
- - - • Stimulation is independent of
HCII / DS / Thrombin exosite II
Exo I Template mechanism
N
HO- S • Heparin serves as a bridge between
R
antithrombin and exosite II of
Exo II
C thrombin
-
- - - - - • Stimulation is independent of
AT / Hep / Thrombin exosite I
Biochemical Evidence
Deletion of “hirudin-like” domain 1
N
1
2
HO- S
L
10,000 X
C
- - -
N
2 Exosite I
L
HO- S ~5 X
- - -
Dermatan sulfate
Biochemical Evidence
Mutation of thrombin exosite I
N
1
2
HO- S
L
10,000 X
C
- - -
N
1
2 Exosite I
L
HO- S ~5 X
- - -
Dermatan sulfate
Crystallographic Evidence
(Jim Huntington’s thrombin(S195A)-HCII complex)
N
1
2
Exosite I
HO- S
L
C
HCII Thrombin
“Hirudin-like” domain 1 of HCII is bound to exosite I of
thrombin when thrombin interacts with the P1 leucine residue
to form the initial Michaelis complex (before cleavage of the
reactive center loop)
Glycosaminoglycan-binding Sites
444
L
468 105 (“hirudin-like” domains)
Dermatan 189 185
Sulfate R K
193 R R R
192 184
173 Heparin
K
Modeled on ovalbumin
• HCII interacts “non-specifically” with heparin
oligosaccharides
• HCII binds to (and is activated by) a specific structure
within the dermatan sulfate polymer
CH2OH CH2OH CH2OH
O -O SO O O -O SO O O -O SO O
COO- 3 COO- 3 COO- 3
O O O O O O O
OH OH OH
OSO3- NHAc OSO3- NHAc OSO3- NHAc
Iduronic N-acetyl Iduronic N-acetyl Iduronic N-acetyl
Acid Galactosamine Acid Galactosamine Acid Galactosamine
2-sulfate 4-sulfate 2-sulfate 4-sulfate 2-sulfate 4-sulfate
• HCII interacts “non-specifically” with heparin
oligosaccharides
• HCII binds to (and is activated by) a specific structure
within the dermatan sulfate polymer
CH2OH CH2OH CH2OH
O -O SO O O -O SO O O -O SO O
COO- 3 COO- 3 COO- 3
O O O O O O O
OH OH OH
OSO3- NHAc OSO3- NHAc OSO3- NHAc
Iduronic N-acetyl Iduronic N-acetyl Iduronic N-acetyl
Acid Galactosamine Acid Galactosamine Acid Galactosamine
2-sulfate 4-sulfate 2-sulfate 4-sulfate 2-sulfate 4-sulfate
Active
CH2OSO3- CH2OSO3- CH2OSO3-
O HO O O HO O O HO O
O COO- COO- COO-
OH O O OH O O OH O O
OSO3- NHAc OSO3- NHAc OSO3- NHAc
Iduronic N-acetyl Iduronic N-acetyl Iduronic N-acetyl
Acid Galactosamine Acid Galactosamine Acid Galactosamine
2-sulfate 6-sulfate 2-sulfate 6-sulfate 2-sulfate 6-sulfate
Inactive
• HCII interacts “non-specifically” with heparin
oligosaccharides
• HCII binds to (and is activated by) a specific structure
within the dermatan sulfate polymer
CH2OH CH2OH CH2OH
O -O SO O O -O SO O O -O SO O
COO- 3 COO- 3 COO- 3
O O O O O O O
OH OH OH
OSO3- NHAc OSO3- NHAc OSO3- NHAc
Iduronic N-acetyl Iduronic N-acetyl Iduronic N-acetyl
Acid Galactosamine Acid Galactosamine Acid Galactosamine
2-sulfate 4-sulfate 2-sulfate 4-sulfate 2-sulfate 4-sulfate
• The distribution of GAGs with high affinity for HCII in
various tissues is currently unknown
• These GAGs probably determine the sites of thrombin
inhibition by HCII under normal or pathologic conditions
Model for Stimulation of HCII by
Dermatan Sulfate
Exosite I
L
GAG-binding site
C
HO- S 1X
N
“Hirudin-like”
1 2 (~5 x 104 M-1min-1)
domain HCII Thrombin
N
1
2
HO- S
L
10,000 X
C
- - - (~5 x 108 M-1min-1)
Dermatan sulfate
Biochemical Evidence
Deletion of the N-terminal “hirudin-like” domain of HCII
L
GAG-binding site Lower affinity for
C
N heparin-agarose
1 2
“Hirudin-like”
domain
L Higher affinity for
C heparin-agarose
N
Biochemical Evidence
Mutation of “hirudin-like” domain 2
L
GAG-binding site HO- S 1X
C
N
1 2
“Hirudin-like”
domain
L
HO- S
C
N
1 2
Biochemical Evidence
Mutation of “hirudin-like” domain 2
L
GAG-binding site HO- S 1X
C
N
1 2
“Hirudin-like”
domain
N
1
2
HO- S
L
~1000 X
C
constitutively
active
No GAG present
Biochemical Evidence
Mutation of “hirudin-like” domain 2
L
GAG-binding site HO- S 1X
C
N
1 2
“Hirudin-like”
domain
N
1
2
HO- S
L
~1000 X
C
constitutively
1 2 active
No GAG present
Biochemical Evidence
Mutation of the GAG-binding site
L
GAG-binding site HO- S 1X
C
N
1 2
“Hirudin-like”
domain
L
HO- S
C
N
1 2
Biochemical Evidence
Mutation of the GAG-binding site
L
GAG-binding site HO- S 1X
C
N
1 2
“Hirudin-like”
domain
N
1
2
HO- S
L
~1000 X
C
constitutively
active
No GAG present
Biochemical Evidence
Mutation of the GAG-binding site
L
GAG-binding site HO- S 1X
C
N
1 2
“Hirudin-like”
domain
N
1
2
HO- S
L
~1000 X
C
constitutively
1 2 active
No GAG present
Structure of an HCII Dimer
(Jim Huntington)
2
Crystal
Acidic GAG- binding Reactive
Domain Domain Site
1 53 75 173 193 444 480
P1 Leu
1 2 Native HCII
“hirudin-like”
Structure of an HCII Dimer
(with apologies to Jim Huntington)
GAG-binding site
(monomer 1)
juxtaposed part of
reactive center 2 “hirudin-like” domain 2
loops (monomer 2)
GAG-binding site
(monomer 2)
Alternative Models for Stimulation
of HCII by Dermatan Sulfate
N
1
2
L HO- S
L
C
N C
1 2
- - -
N
N 1
2
1
2 HO- S
L L
C C
- - -
What is the Physiologic Function of HCII?
(1) Synthesized in the liver
Plasma concentration ~1 µM
Half-life ~2.5 days
Extravascular distribution ~40%
(2) Thrombin-HCII complexes are found
in normal plasma
(suggests that HCII inhibits
thrombin in vivo)
(3) Heterozygous HCII deficiency (~50%)
in ~1% of normal blood donors
~1% of patients with venous
thrombosis
Homozygous (?) HCII deficiency (10-
15%) reported in 2 sisters, one with
thrombosis
(4) Administration of dermatan sulfate
has antithrombotic activity in animals
and humans
(5) Elevated levels of HCII (~150%) and
thrombin-HCII complex (~400%) in
pregnant women at term
(6) Acquired HCII deficiency (~50%) in
women with severe pre-eclampsia
(7) Dermatan sulfate proteoglycan
(decorin) with HCII stimulating
activity isolated from human placenta
(8) HCII is present in the intima of normal
human arteries
(9) Atherosclerotic lesions have altered
dermatan sulfate proteoglycans with
decreased HCII stimulating activity
(10) Potential roles of thrombin in
inflammation (chemotaxis) or wound
healing (mitogenesis)
HCII Is Conserved Among Vertebrates
Human MKHSLNALLIFLIITSAWGGSKGPLDQLEKGGETAQSADPQWEQLNNKNLSMPLLPADFHKENTVTNDWIPEGEEDDDYLDLEKIFSEDDDYIDIVDSLSVSPTDSDVSAGNILQLFHGK 101
-19
Rabbit MQHRPHLLLISLTIMSVCGGSNG-LT----------------DQLNNKNLTMPLLPIEFHKENTVTNDWIPEGEEDDDYLDLEKLLSEDDDYIDIID--AVSPTDSEASAGNILQLFQGK 82
-19
Rat -18 MKHPAYTLLLSL-IMSMCAGSKGLA-----------------EQLTKENLTVSLLPPNFHKENTVTNDWIPEGEEDDDYLDLEKLLSEDDDYIYVVD--AVSPTDSESSAGNILQLFQGK 82
Mouse MKHPLCTLLSLITFMCI--GSKGLA-----------------EQLTNENLTTSFLPANFHKENTVTNDWIPEGEEDEDYLDLEKLLGEDDDYIYIID--AVSPTDSESSAGNILQLFQGK 76
-23
Frog -18 MKLLHLAT-IFLLIHATLGGVKDLQEHFE---DTSTGINPRGSQ--TQAVE-NLL------DDTVTNDLSTEGEDEEDYLDFDKIFGEDEDYIDIIDA-APEIKNSETQQGNIFELFHGK 88
Chicken
1 GTFCGIKDFSDHF----ESLK--DAH-TH-ENGTYNMPDLPLEFHRENTITNDLIPEEEEEEDYLDLDKILGED-DYSDIID--AAPHIVSEIQQGNILELFQGK 94
“hirudin-like” domain
Human SRIQRLNILNAKFAFNLYRVLKDQVNTFDNIFIAPVGISTAMGMISLGLKGETHEQVHSILHFKDFVNASSKYEITTIHNLFRKLTHRLFRRNFGYTLRSVNDLYIQKQFPILLDFRTKV 221
102
Rabbit83 SRIQRLNILNAKFAFSLYRALKDQANAFDNIFIAPVGISTAMGMISLGLKGETHEQVHSVLHFRDFVNASSKYEILTIHNLFRKLTHRLFRRNFGYTLRSVNDLYVQKQFPIREDFKAKV 202
Rat 83 SRIQRLNILNAKFAFNLYRVLKDQATSSDNIFIAPVGISTAMGMISLGLRGETHEEVHSVLHFKDFVNASSKYEVTTIHNLFRKLTHRLFRRNFGYTLQSVNDLYIQKQFPIREDFKAAM 202
Mouse77 SRIQRLNILNAKFAFNLYRVLKDQATTSDNLFIAPVGISTAMGMISLGLRGETHEEVHSVLHFRDFVNASSKYEVTTIHNLFRKLTHRLFRRNFGYTLRSVNGLYIQKQFPIREDFKAAM 196
Frog 89 TRVQRLNIINANFGFNLYRAIKNNTDASENILLAPVGISTAMATISLGTKGQTLEQVLLTLGFKDFLNASSKYEILTLHNVFRKLTHRLFRRNFGYTLRSVNDIYVKRDFLIREPFKNNL 208
Chicken TRIQRLNILNANFGFNLYRSVADKANSSDNILMAPVGISTAMAMISLGLKGQTQQEVLSVLGFEDFINASAKYELMTVHNLFRKLTHRLFRRNFGYTLRSVNDLYIRKDFSILNDFRNNM 214
95
GAG-binding site
Human REYYFAEAQIADFSDPAFISKTNNHIMKLTKGLIKDALENIDPATQMMILNCIYFKGSWVNKFPVEMTHNHNFRLNEREVVKVSMMQTKGNFLAANDQELDCDILQLEYVGGISMLIVVP 341
222
Rabbit REYYFAEAQAADFSDPAFISKANNHILKVTKGLIKEALENVDPATQMMILNCIYFKGTWVNKFPVEMTHNHNFRLNEREVVKVSMMQTKGNFLAANDQELACDVLQLEYVGGISMLIVVP 32
203
Rat 203 REFYFAEAQEADFSDPAFISKANSHILKLTKGLIKEALENTDSATQMMILNCIYFKGAWMNKFPVEMTHNHNFRLNEREVVKVSMMQTKGNFLAANDQELDCDILQLEYVGGISMLIVIP 322
Mouse REFYFAEAQEANFPDPAFISKANNHILKLTKGLIKEALENIDPATQMLILNCIYFKGTWVNKFPVEMTHNHNFRLNEREVVKVSMMQTKGNFLAANDQELDCDILQLEYVGGISMLIVVP 316
197
Frog 209 KNYYFAEAQTVDFGYKDFLTKANKRIQQLTKGLIEEALTNVDPALLMLLVNCIYFKGTWENKFPVEYTQNMNFRLNEKELVKVPMMKTKGNFLVAADPELDCAVLQLPYVGNISMLIVLP 32
Chicken KTYYFADAQPADFSDPNFITKTNERILKLTKGLIKEALVNVNPTTLMMILNCLYFKGTWENKFPVEMTTKRSFRLNEKQTIKVPMMQTKGNFLAAADPELDCGVIQLPFVGNISMLIVLP 334
215
Human HKMSGMKTLEAQLTPRVVERWQKSMTNRTREVLLPKFKLEKNYNLVESLKLMGIRMLFDKNGNMAGISDQRIAIDLFKHQGTITVNEEGTQATTVTTVGFMPLSTQVRFTVDRPFLFLIY 461
342
Rabbit HKLSGMKTLEAQLTPQVVERWQKSMTNRTREVLLPKFKLEKNYNLVEALKSMGVTELFDKNGNMSGISDQGITMDLFKHQGTITVNEEGTQAAAVTTVGFMPLSTQVRFTVDRPFLFLVY 4
323
Rat 323 RKLSGMKTLEAQLTPQVVERWQKSMTNRTREVLLPKFKLEKNYNLVEVLKSMGITKLFNKNGNMSGISDQRIIIDLFKHQSTITVNEEGTQAAAVTTVGFMPLSTQVRFTVDRPFLFLVY 442
Mouse RKLSGMKTLEAQLTPQVVERWQKSMTNRTREVLLPKFKLEKNYNLVEVLKSMGITKLFNKNGNMSGISDQRIAIDLFKHQSTITVNEEGTQAAAVTTVGFMPLSTQVRFTVDRPFLFLVY 43
317
Frog 329 HKLSGMKLLEKQISPQVVERWQNIMTNRTREVFLPRFKLEKSYDLQKVLSNMGATDLFT-HGDFSGVSDKDINIGLFQHQGTITVNEEGTEAAAVTVVGFMPLSTQARFVADRPFLFLIY 447
Chicken HKLSGMKALEKQITPQVVEKWQKSMTNRTREVVLPKFKLEKNYNLIGFLRSMGIEELFSEKGNYCGVSEEKVSIDRFNHQGTITVNEEGTEAGAITNVGFMPLSTQIRFIVDRPFLFLIY 454
335
P1 residue
Human EHRTSCLLFMGRVANPSRS 480
462
Rabbit EHRTSCLLFMGKVANPVRS 461
443
Rat 443 EHRTSCLLFMGRVANPAKS 461
Mouse EHRTSCLLFMGKVTNPAKS 455
437
Frog 448 EHRTNCLVFMGRVANPTKS 466
Chicken EHRTNCLLFMGRVVNPAKP 473
455 Human 85.0 83.0 82.8 58.9 65.3 % identity
Knockout Strategy
14 kb
BamHI
EcoRI
exon exon exon exon
SacI
SacI
1 2 3 4
Wild-type
HCII locus
BamHI
EcoRI
SacI
Targeting
neo
vector
BamHI
EcoRI
SacI
SacI
SacI
Targeted
neo
HCII locus
probe
6 kb
Generation of HCII-deficient Mice
Offspring
# %
HCII+/+ 39 25.3
HCII+/- x HCII+/- HCII+/- 78 50.6
(F1 cross) HCII-/- 37 24.0
154
14 kb
6 kb
Genotype -/- +/+
Western Blot
72 kDa
68 kDa
+/+ +/– –/– +/+ +/– –/– HCII Genotype
0.1 µl 0.3 µl
Mouse Plasma
Plasma Activity Assays
Heparin Cofactor II Antithrombin
Mouse plasma (2 µl) Mouse plasma (0.3 µl)
Dermatan sulfate (50 µg/ml) Heparin (7 µg/ml)
Human thrombin (16 nM) Human factor Xa (11 nM)
AT Activity (µM)
3.00
HCII Activity (µM)
0.45 n = 11 n = 11 n=9 n = 10
2.50
0.35
2.00
0.25 n=9 1.50
0.15 1.00
n = 10 0.50
0.05
0.00
-0.05 +/+ +/– –/– +/+ +/– –/–
Genotype Genotype
Growth of HCII-/- Mice
50 Males
45
+/+ (n=5)
+/- (n=4)
40 -/- (n=5)
Weight (g)
Weight (g)
35 Females
+/+ (n=8)
30
+/- (n=10)
25 -/- (n=6)
20
mean + S D
15
61- 71- 81- 91- 101- 111- 121- 131- 141- 151- 161- 171- 181-
70 80 90 100 110 120 130 140 150 160 170 180 190
Age (d)
Blood Tests
HCII+/+ HCII-/-
WBC 6.4 + 1.3 6.3 + 2.2
seg 19 + 8 25 + 2
band 0 + 0 1 + 1
lymph 80 + 8 73 + 22
mono 1 + 1 2 + 1
eos 0 + 0 0 + 0
baso 0 + 0 0 + 0
RBC 11.1 + 0.4 10.2 + 1.6
Hgb 16.8 + 0.8 16.1 + 2.4
MCV 49.2 + 1.7 50.8 + 1.3
Platelets 1209 + 142 1031 + 279
BUN 23 + 3 24 + 8
Creatinine 0.2 + 0.1 0.3 + 0.1
Total protein 5.2 + 0.3 5.1 + 0.5
ALT 80 + 83 101 + 97
AST 203 + 190 235 + 118
Fertility of HCII-/- Mice
(mean + SD)
10
8
Litter Size
6
4
2
0
-/- X -/- +/- X +/-
Cross
Phenotype of Unchallenged HCII-/- Mice
(1) Normal fetal development
(2) Normal growth to 1 year of age
(3) No morphologic abnormalities on
gross or microscopic examination
(4) No liver, kidney, or blood cell
abnormalities
(5) Fertile
Phenotype of Unchallenged HCII-/- Mice
(1) Normal fetal development
(2) Normal growth to 1 year of age
(3) No morphologic abnormalities on
gross or microscopic examination
(4) No liver, kidney, or blood cell
abnormalities
(5) Fertile
(6) Lack dermatan sulfate-dependent
thrombin inhibition in plasma
Phenotype of Challenged HCII-/- Mice?
(1) Arterial injury
(2) Venous injury
(3) Inflammation
(4) Wound healing
(5) Bleeding
(6) Tumor metastasis
(7) Atherogenesis (x apoE-/-)
(8) Thrombogenesis (x factor VLeiden)
Carotid Artery Thrombosis Model
1. Isolate common carotid artery
Carotid Artery Thrombosis Model
1. Isolate common carotid artery
2. Apply ultrasonic flow probe
Carotid Artery Thrombosis Model
1. Isolate common carotid artery 3. Illuminate with 540 nm laser
2. Apply ultrasonic flow probe
Carotid Artery Thrombosis Model
1. Isolate common carotid artery 3. Illuminate with 540 nm laser
4. Inject rose bengal dye IV
at time = 0
(photochemical reaction
generates singlet oxygen
within the arterial lumen)
2. Apply ultrasonic flow probe
Carotid Artery Thrombosis Model
1. Isolate common carotid artery 3. Illuminate with 540 nm laser
4. Inject rose bengal dye IV
at time = 0
(photochemical reaction
generates singlet oxygen
within the arterial lumen)
5. Measure time to complete
occlusion
2. Apply ultrasonic flow probe
Carotid Artery Thrombosis Model
Rose Bengal IV Occlusion Time
Flow (ml/min)
1.0
0.5
0.0
0 5 10 15 20 25 30 35
Time (min)
Occlusion Time After
Endothelial Injury
30
Occlusion Time (min)
25
(mean + SD)
p = 0.012
20
15
10
5
0
+/+ –/–
HCII Genotype
Questions Raised by the
Thrombosis Experiments
Does the protective effect of HCII
depend on activation by GAGs in the
vessel wall?
Is the effect similar in veins and
arteries?
Hypothetical Interactions of HCII and
Antithrombin with Vascular Proteoglycans
Endothelial Cells
AT bound to HS
proteoglycan
HCII bound to DS or HS
proteoglycan
Fibroblasts / Smooth Muscle Cells
Stimulation of HCII by
IMR-90 Human Fetal Lung Fibroblasts
125I-thrombin
+ 125I-thrombin-2M
plasma
125I-thrombin-HCII
125I-thrombin-AT
30 min
SDS-PAGE
autoradiography
125I-thrombin
Cells: + –
Stimulation of HCII by
Cell Monolayers
Human Fetal Lung Fibroblasts +
Human Adult Skin Fibroblasts +
Porcine Aortic Smooth Muscle Cells +
Human Umbilical Vein Endothelial Cells –
Murine Macrophages –
Question
Does the protective effect of HCII
depend on activation by GAGs in the
vessel wall?
Experimental Approach
Reconstitute HCII-/- mice with wild-type
recombinant HCII or mutants with
defects in binding to dermatan sulfate
or heparan sulfate
Conclusions
(1) Unchallenged HCII-deficient mice
have an apparently normal phenotype
(2) HCII inhibits thrombosis of the mouse
carotid artery after endothelial injury
Conclusions
(1) Unchallenged HCII-deficient mice
have an apparently normal phenotype
(2) HCII inhibits thrombosis of the mouse
carotid artery after endothelial injury
(3) Do abnormalities of HCII play a role in
human arterial disease?
Investigators
Washington University University of Michigan
Morey Blinder Dan Eitzman
Guangsen Zhang Randy Westrick
Niall Colwell
Betsy McGuire
Mauro Pavao
Li He
Margaret Maimone
Vivianna Van Deerlin
Jin-Hua Han
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