Blood Clotting

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Blood Clotting
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Major Components of Blood Clotting
1) Intact Endothelial Cells - inhibit blood
clotting - surface not conducive to clot
formation - display membrane proteins that
inhibit clotting - store von Willebrand
factor in cytop. granules. Constituitively
expressed and secreted into circulation
or subendothelium. (20 made by platelets,
rest endothelium) - make prostacyclin
inhibits platelet aggregation
Endothelial cells
http//www.meddean.luc.edu/lumen/MedEd/orfpath/mur
ali.htm
http//www.vet.uga.edu/vpp/CLERK/anderson/
Endothelial cells with stored vWF (red)
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2) Subendothelial Cells - contain membrane prots
and extracellular matrix prots (collagen) that
normally do not contact blood - When exposed
after injury, platelets aggregate at the site by
mediation of von Willebrand factor (vWF) that
binds to both platelet receptors and
collagen/subendo cells - vWF is a large,
multimeric protein with subunits of 225 kD each
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3) Platelets - unpigmented, enucleated cells
that are fragments of larger progenitor cells
called megakarocytes (bone marrow). - Once
bound release TxA2 and seratonin (and more!) that
induce vasoconstriction to reduce blood flow
and increase platelet aggregation.
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Platelets
Platelets bleb off of megakarocytes
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4) Clotting Factors - Soluble plasma
proteins - most made in liver - most are
serine proteases and circulate as
zymogens - cascade in which clotting factors
are activated by selective proteolytic
cleavage must have Ca2
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Factor Common Name Number I Fibrinogen II
Prothrombin III Tissue Factor IV Ca2 Va
Proaccelerin VII Proconvertin VIII Antihemophi
lic Factor IX Christmas Factor X Stuart
Factor XI Plasma thromboplastin
antecedent XII Hageman factor XIII Fibrin
Stabilizing Factor
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Step 1 Platelet Aggregation
? Platelet adhesion is mediated by vWF. This
activates platelets causing release of TxA2 ?
During activation, a receptor for fibrinogen
becomes exposed on the platelet membrane. ?
Activated platelets release Fibrinogen ADP/AT
P vWF Seratonin Factor V Ca2 Factor
VIII Platelet derived growth factor (PDGF)
promotes healing Platelet factor IV prevents
formation of active thrombin inhibitor from
heparin and anti-thrombin III.
dense core granules
a-granules
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• Platelet activation also induces large
  morphological changes
• - membrane lipids rearrange
• phosphatidyl serine which is usually on the
• inner membrane of the platelet,
• flips out to outer membrane where it plays a role
  
• in binding prothrombin.

phosphatidyl serine
unactivated
fully activated
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Clotting Pathways
1) Intrinsic all components are in the
blood - must have a negative surface (glass
slide) 2) Extrinsic at least one component
from tissue injury response - need tissue
factor (TF) for activation of VIIa - TF and
factor VIIa contact only after injury -
TF-factor VIIa complex also activates factor
XIa - Clotting is localized by requiring a
negatively charged surface so initially,
platelets bind collagen, and later clot
formation promoted by platelet membranes on
surface of clot.
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Step 2 Fibrin is formed by cleaving fibrinogen
(factor I)

• Thrombin catalyzes the conversion of fibrinogen
  to fibrin
• - Fibrinogen is 2-3 of plasma protein
• - Fibrinogen has 3 pairs of non-identical
  subunits
• Aa (610 res) Bb (461 res) g (411 res)
• Also two pairs of N-linked oligosaccharides
• A and B refer to parts of a and b released
• upon thrombin cleav. A (20 aa) B (18 aa)

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• Once cleaved, fibrin is insoluble
• So how do A and B confer solubility?
• A B mask sites that mediate
• aggregation
• 2) A B are highly anionic and
• so repel other fibrinogen molecules

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Step 3 Conversion of soft fibrin clots to hard
clots

• Involves crosslinking of neighboring
• fibrin molecules
• Catalyzed by fibrin-stabilizing factor
• (fsf or factor XIIIa)
• Joins C-term segments of g chains by
• forming isopeptide bonds between Gln
• on one g and a Lys on the other.

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• a are linked also but slower
• If decreased FSF, have increased bleeding
• FSF in both platelets and plasma occur as
  zymogen activated by thrombin
• Thrombin cleaves an Arg-Gly bond near the
  N-terminus of FSF

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FSF Structure Overview

• FSF from platelets has 2 a chains while plasma
• FSF has a2b2
• - Cleavage by thrombin activates platelet FSF
  (a2)
• but plasma FSF (a2b2) remains inactive
• until its b chains dissociate
• Dissociation is triggered by Ca 2 binding to
  the a subunits
• Ca 2 is essential for clotting, released by
  platelets
• - b subunits inhibit activation in absence of Ca
  2 and prolong
• FSF lifetime in plasma

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FSF Activation
Platelet FSF
Thrombin (tbn) cleaves FSF at its N-term end b
subunits dissociate when Ca binds, then tbn
activates FSF
a
a
tbn
a
a
inactive
active
Plasma FSF
Ca
Ca
Ca
a
a
a
a
tbn
b
b
Ca
a
a
a
a
b
b
b
Ca
Ca
Ca
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Overview of Thrombin Activation
Thrombin needs to active only locally, at site of
injury Circulates in plasma as prothrombin
single chain of 582 aa (almost twice the size of
active thrombin) Monomer in zymogen but a dimer
when active A chain (36aa) and B chain
(259aa) connected by disulfide bond Prothrombin
is cleaved by factor X twice Arg271 Thr
272 releases N-term propeptide Arg320 Ile
321 separates A and B chains Second cleavage
activates, allowing an ion pair NH4 of Ile321
and Asp524
X
propep
A
B
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Propeptide of Prothrombin
? Propeptide has 3 domains 1) N-term 40 residue
Gla domain - Gla g carboxyglutamate - Strong
Ca2 chelator - Bind Ca 2 that mediates
interaction with phospholipid membrane of
platelet
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Vitamin K as a Cofactor

• - Vit K is essential cofactor for
• proper prothrombin synthesis
• - Must have in diet or no clotting
• - Prothrombin is made in liver
• - Made without Vit K but only 1-2
• active. Reason is that Vit K
• is a necessary cofactor for
• post-translational modification of
• prothrombin in ER
• Needed for Glu conversion to Gla

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Vitamin K cofactor in Glu to Gla conversion

• Conversion of Glu to Gla
• Vitamin K extracts a g proton from Glu, yielding
  a carbanion
• 2) The carbanion then reacts
• with CO2 to yield Gla
• 3 4) Vitamin K is
• regenerated

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Inhibitors of Vitamin K Regeneration
? Take awhile to take effect because turnover
of coagulation proteins is relatively slow
(5-7 days). ? Wont affect prots synthesized
before ingestion
warfarin (rat poison)
dicoumarol (spoiled sweet clover)
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Pro segment of Thrombin
Green Kringle Orange Gla domain Blue
Calcium ions Red Sticks -- Gla
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Val34Leu Mutation

Val34Leu
Wild Type
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FSF Domains
Activation peptide (green/cyan) 1-37 b-sandwich
1 (yellow) 38-185 Catalytic domain (orange)
186-503 b-barrel 1 (violet) 517-628 b-barrel 2
(red) 632-731
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FSF needs Ca2 for activation
Active FSF binds 3Ca2
Inactive FSF binds 1Ca2
EMBO (2002) 21, 9, 2055-2067.
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EMBO (2002) 21, 9, 2055-2067.
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EMBO (2002) 21, 9, 2055-2067.
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vWF - GpIba structure
vWF has 3 domains A1 binds to GpIba receptor on
platelet A2 proteolytic cleavage domain
(degradation) A3 binds to exposed collagen in
subendothelium GpIb has 4 transmembrane
subunits GpIX, GpIV, GpIba, GpIbb
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vWF Disease
Type 2B vWF disease is caused by a qualitative
abnormality of vWF Increased affinity of vWF
for GpIba Normally, vWF only binds to platelets
under high sheer conditions Results in increased
bleeding because all vWF tied up with
platelets Majority of mutations occur between
Cys509-Cys695 Also mutation in GpIba also have
higher affinity for vWF Called platelet-type vWD
and has same result involves Met239Val
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Anthrax
Anthrax toxin is composed of three non-toxic
proteins (1) Protective Antigen (PA) (2)
Edema Factor (EF) (3) Lethal Factor
(LF) Released from anthrax as inactive monomers
that assemble into toxic complexes on the
surface of host cells Full length PA binds to a
receptor homologous to vWF cleaved and the
remaining PA oligomerizes into a
heptameric receptor bound prepore Oligomerizatio
n creates binding sites for 3 EF and/or LF
Anthrax
PA
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Anthrax (cont.)
? The entire toxin-receptor complex is
endocytosed and trafficked to a low-pH
endosome ? The high acidity of the environment
causes a conformational rearrangement of the PA
assembly allowing the formation of a pore across
the endosomal membrane ? EF and/or LF is then
released into the cytosol through this pore ? EF
is an adenylate cyclase makes cAMP which
impairs macrophages ? LF is a zinc protease
that cleaves MAP kinases which impairs
dendritic cell function and induces apoptosis in
macrophages and endothelial cells
EF
LF
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Infection of Anthrax
PA
PA
LF
LF
EF
EF
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Blood Clotting Disorder Assignment
Set A AFIBRINOGENEMIA, CONGENITAL ROSENTHAL
SYNDROME THROMBOPHILIA, HEREDITARY, DUE TO
DEFICIENCY OF AT-III and PC FAMILIAL MULTIPLE
COAGULATION FACTOR DEFICIENCY I Set
B DYSFIBRINOGENEMIA HAGEMAN FACTOR DEFICIENCY VON
WILLEBRAND DISEASE LABILE FACTOR DEFICIENCY (cf.
Quebec platelet disorder and APC resistance) Set
C FACTOR XIII DEFICIENCY BERNARD-SOULIER SYNDROME
(type A,B,C) HYPOPROTHROMBINEMIA AND
DYSPROTHROMBINEMIA FAMILIAL MULTIPLE COAGULATION
FACTOR DEFICIENCY II Set D HYPOPROCONVERTINEMIA HE
MOPHILIA A and B STUART-PROWER FACTOR DEFICIENCY
(why called this?) PROTEIN S Deficiency