Transcript Thrombocytes/ Platelets

Hemostasis: stopping the flow of blood
Thrombocytes = Platelets
Chap 36
1
• Thrombocytes = platelets
• They need homeostasis of their hemostasis
(cessation of bleeding). You want to stop
bleeding, but not excessive clotting.
•
• Too much clotting, get thrombus or
embolis.
• Too little, get hemorrhaging.
NORMAL HAEMOSTATIC BALANCE
Pro-coagulants
and platelets.
Thrombosis
(inappropriate clotting)
Naturally
occurring
anticoagulants and
good vascular flow
(normally
dominant)
Bleeding
Failure to clot
By the end of this class:
3
You should be able to name naturally occurring coagulants and anti-coagulants
• You have thousands of micro-tears in your vasculature
every day. Platelets clot them constantly.
• They come from a stem cell, hemocytoblast, from the
myeloid stem cell line. It comes from a megakaryocyte,
which is a differentiated cell. It sheds off some cytoplasm
and pinches off a piece of membrane to become a
platelet. Granules inside activated platelets are released,
activating more platelets (positive feedback). An activated
platelet changes from disc to spiny shape, like a sea
urchin. When all clotting factors are consumed and there
is still bleeding, this is disseminated intravascular
coagulation (DIC), usually what causes death during baby
delivery. There is an ultimate negative feedback to stop it.
Platelets
• Platelets = thrombocytes (thrombus=clot)
• Life span 8-12 days
• Contain intracellular granules (α and ) that contain coagulation
factors and ADP.
• Actin, myosin (important contractile proteins)
• Activated platelets form temporary plug.
• On average there are 150200,000 platelets/µL of
blood.
• Produced in the bone
marrow from
megakaryocytes.
• Production stimulated by
thrombopoietin from
liver/kidney
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Genesis of Blood Products
T-Cell
CFU-T
B-Cell
CFU-B
Pluripotent
Stem Cell
hemocytoblast
Lymphoid
Stem Cell
CFU-L
eosinophil
CFU-Eosin
basophil
CFU-Bas
neutrophil
CFU-GM
Myeloid
Stem Cell
CFU-M
Copyright © 2006 by Elsevier, Inc.
monocyte
macrophage
platelets
CFU-MEG
BFU-E
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erythrocyte
• ADP in the granules is one of the things that activate more
platelets. Other things that activate them are collagen
from under the endothelial cells, smoking, high estrogen
levels (from BCP). There are 150-200K platelets per µl of
blood. Platelets also have actin and myosin. When the clot
is made, the actin and myosin interact with each other,
pulls the ends of the blood vessel together.
•
• Thrombopoeitin is released in liver and kidney to stimulate
production of platelets.
Main phases of hemostasis
• Vasoconstriction: artery is cut, both ends
constrict
• Platelet plug: severed ends exposed to
collagen, activates platelets, ADP released,
pile on top of each other, into an unstable
platelet plug.
• Coagulation: fibrin polymers weave around
each other to form clot
• Clot retraction: pulling cut end together
• Clot dissolution
• Tissue repair
Hemostasis
•
•
•
Prevents blood loss thru the walls of damaged
blood vessels
Also establishes a framework for further tissue
repairs
6 main phases:
1. Vasoconstriction
2. Platelet plug formation
3. Coagulation (clot formation)
4. Clot retraction
5. Clot dissolution (fibrinolysis)
6. Tissue repair
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VASCULAR PHASE
• The smooth muscle constricts, less blood spills out. How
much blood is lost depends on the radius of the blood
vessel, so it is important to apply pressure to a cut. Some
chemicals increase vasoconstriction, such as
thromboxane A2 (released by damaged endothelium),
prostaglandin 2 (PG2). Thromboxane A2 is released by
activated platelets, and it also acts to further activate more
platelets (Positive feedback). Cyclooxygenase (COX-1 )
blocks arachidonic acid, which blocks production of
thromboxane A2. Aspirin blocks COX-1 and you lose
vasoconstriction ability.
Blood vessel damage
• Smooth muscle in blood vessel wall
contracts (vascular spasm)
• Blood vessel diameter decreases
• Blood loss slows
• Myogenic contraction of smooth muscle
• Enhanced by release of thromboxane A2
(vasoconstrictor) and local myogenic
spasm, local factors, and nervous reflexes
• Narrows damaged vessels
Blood Vessel Damage
Smooth muscle in BV wall
contracts – vascular spasm
BV diameter 
Blood loss slows
Vascular Phase
• Myogenic contraction of
smooth muscle
• Enhanced by release of
thromboxane A2
(vasoconstrictor) and
local myogenic spasm,
local factors, and nervous
reflexes
• Narrows damaged
vessels
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PLATELET PLUG
• Platelets are normally disc shaped, but activated ones
change shape and become spiny all over, exposing the
receptors on them. Von Willebrand factor helps the
platelet receptors anchor to the torn blood vessel. Inside
the platelet, the granules release their contents, including
ADP, which is dumped into the plasma. ADP is the #1
signal to recruit more platelets. Calcium, serotonin,
thromboxane A2 are also released. As the platelets are
activated and sticky, they will stick to exposed collagen
and to each other in a platelet plug. It is not permanently
fixed in place, that’s the clotting phase. In this stage, the
platelet plug is unstable, and BP could dislodge it.
2. Platelet Phase
• Platelets are normally repelled from the blood vessel endothelial lining
• When trauma occurs, traumatized platelets lyse and release their granular
contents ADP and thromboxane A2. Occurs within 15sec of the injury
• Platelets attach to sticky endothelial cells, the basement membrane, and
to exposed collagen fibers. The endothelial cells release Von Willebrand
factor (vWF also is a carrier for VIII).
• These cause other platelet membranes to become “sticky”, then
release…. (positive feedback) more release, more platelets…
(amplification effect)
– ADP  the primary stimulus for platelet aggregation
– Thromboxane A2  causes platelet aggregation and local
vasoconstriction
– Serotonin  causes local vasoconstriction
– Calcium ions
•
•
Mounds of platelets at damage site forms a plug.
This is NOT a clot! The plug is a weak “thumb in the leaky dam” fix. Can dislodge
and leak again. Still need to form a firm web of fibers within the plug
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15
Factors that are against clotting
• What prevents platelets from becoming
activated? Endothelial cells also release a
different prostaglandin called prostacyclin
and nitric oxide help repel the platelets
from the endothelial cells, so they cannot
activate properly. These are anticoagulants.
Factors that promote clotting
• A tear in vessel, collagen fibers provide a
place for Von Willebrand factors to allow
platelets to bind.
• Just one platelet activated, releases ADP
Thromboxane A2,, serotonin, calcium.
These help clotting.
Let’s go over the activators of
platelets again
Collagen
•
• Activated platelets—
sounds like positive feed
back, yes?
• ADP released from
damaged RBCs and
activated platelets
• Thromboxane A2
• Epinephrine (stress)
• Thrombin— Look for this
again later! Positive
feedback mechanism!
• Smoking
• Estrogen (BCP): don’t
smoke and take these, or
can get a blood clot!
Active platelets change morphology
from tiny discs to spheres
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Platelet Facts
• Thromboxane in platelets is formed from
fatty acid precursor, arachidonic acid with
the enzyme cyclooxygenase (COX-1
enzyme.)
• Aspirin is an irreversible COX enzyme
inhibitor; blocks formation of thromboxane;
platelets don’t stick and plug is less likely to
form. So, aspirin reduces tendency of those
platelets to plug, and thus reduce blood
clotting tendency.
• Often prescribe aspirin to those with greater
tendency to clot, to reduce risk of heart
attack and stroke, (baby aspirin)
• Must wait till exposed platelets are removed,
turnover time, approx 1 wk-10 days
Undergoing surgery? Why is scheduling
important?
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COAGULATION
• Deals with a series of clotting factors numbered 1-13, but there are
only 12 of them!
• The end result is to have fibrin threads create a meshwork; within it
are platelets and RBCs trapped in it. But RBCs have no part in the
clotting process, they just got trapped there. They contribute to the
clot, but don’t make it. The fibrin meshwork makes the clot.
• Calcium and coenzymes help enzymes do their job more efficiently.
• If an enzyme is prefixed with pro-, or ends in -ogen, it is in its inactive
form.
• Prothrombin  thrombin
• Fibrinogen  fibrin
3. Coagulation



It is an amplification pathway (cascade;
positive feedback).
The end result is a clot (thrombus). A
web of fibers (fibrin threads) which
support and make the platelet plug
strong.
Requires clotting factors such as:

Calcium ions.

12 different factors, some synthesized
by the liver


Many are proenzymes or zymogens
(inactive enzymes)
The synthesis of some of them requires
Vitamin K (II, VII, IX, X)
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Common Clotting Factor Names and Numbers
(add a subscript “a” to each when each becomes activated.)
I …………………..…. Fibrinogen
II ……………..…….. Prothrombin
III …………...………. Tissue thromboplastin
IV ………………...…. Calcium
VI………………...…. Doesn’t exist
VIII …………….….... Antihemophilic Factor (composed of 2
fragments: the small VIIIc and larger von Willebrand Factor)
IX ………………….. Christmas Factor (Christmas hemophilia)
XI and XII………….Contact Factors (don’t req calcium)
XIII ……………...….. Fibrin stabilizing factor
Future
Physiologists
take fun classes
5-13 years.
Mnemonic for memorizing the first 7 clotting factors:
Future = fibrinogen
Physiologists = prothrombin
Take fun = tissue factor
Classes = calcium
5 -13 Years = factors 5-13
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3. Coagulation Phase (Clot formation)
• Begins 30 sec or more after
vessel damage occurs
• Involves a sequence of steps
leading to the conversion of
fibrinogen (a circulating plasma
protein) to the insoluble protein
fibrin.
• A network of fibrin grows and
covers the surface of the platelet
plug. RBCs and additional
platelets are trapped in this
tangle, forming a blood clot
(thrombus) that effectively seals
the damaged vessel wall.
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Clotting Pathways
• Extrinsic and intrinsic pathways release clotting factors
that activate other clotting factors…positive feedback. The
two pathways converge on one common pathway that
starts with PAS (prothrombin activated substance). The
extrinsic pathway makes PAS faster, but not as much of it.
The intrinsic pathway is has more enzymes involved, so it
is slower to make PAS, but it makes a lot of it. You don’t
need to memorize the intrinsic and extrinsic pathway, just
know the common pathway.
• The common pathway starts with PAS, and it activates
prothrombin  thrombin  fibrinogen  fibrin
• Factor 13 links the fibers together to stabilize the plug.
• The extrinsic pathway is activated when
there is tissue damage.
• The intrinsic pathway is activated when
there is blood or vessel damage.
• You can’t have one or the other pathway
exclusively. Both will get activated.
Clotting Pathways
• The extrinsic pathway is triggered by damage outside the bloodstream,
ex. surrounding tissue and begins with the release of a tissue factor
(lipoprotein).
• The intrinsic pathway is triggered by damage inside the bloodstream
itself (damaged blood elements and endothelial wall). Platelet factor
(PF-3) is a phospholipid that is released
• Charged surfaces, such as glass or plastic tubing can trigger this pathway.
• Heart and lung bypass?
• The extrinsic and intrinsic pathways begin simultaneously, and
converge at the final common pathway
Extrinsic Path
Final Common Path
Intrinsic Path
PAS
Fibrin polymers
The extrinsic pathway is faster, but produces less product (PAS)
The intrinsic pathway is slower (more steps), but yields more product (PAS)
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slower (2-6
minutes)
many
components
(cascade)
Final Common Pathway
Rapid and
explosive in
nature (15
seconds)
The subscript “a”
after a factor
number indicates
“activated”
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Final Common Pathway
Prothrombin Activator Substance (PAS)
Prothrombin
Thrombin
Fibrinogen
Fibrin monomers
Fibrin Stabilizing Factor
It ten plus 5, 4 and 3 equals
(twenty) TWO, then 1+13 equals
glue!
Polymers, Fibrin Threads
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• What factors come together to make PAS?
10, 5, 4, 3
• Mnemonic to remember this:
• If 10 + 5 + 4 + 3 = (twenty) TWO, then 1+13
equals glue!
• 2 = prothrombin (PAS activates factor 2,
which activates thrombin.
• 1 = fibrinogen
• 13 = fibrin stabilizing factor
What does fibrinogen look like?
• Fibrinogen has long fibers with a protein
cap. If bacteria cut off the cap, the exposed
fibrin heads have a negative charge. Their
tail ends have a positive charge. Therefore,
cut fibrinogen fibers will suddenly line up,
head to tail, in a long series of fibers,
making an inefficient clot. The bacteria that
can do this can cause a person to bleed out
(DIC).
What does fibrinogen look like?




6 polypeptide chain with a “cap” on the ends
Thrombin cuts cap off and creates fibrin
Fibrin ends have negative charge and this allows them to align and polymerize
Factor 13 needs to covalently link them together to make a stable fibrin thread
Thrombin chops
these ends off
-
Fibrin monomer
(single monomer)
-
-
Factor
thirteen
links fibrin
monomers
-
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4. Clot Retraction
• Once the fibrin meshwork has appeared, RBCs &
platelets stick to the fibrin strands.
• Clot retraction (30-60 minutes)
• The platelets then contract and the clot undergoes clot
retraction which:
– Pulls the torn edges of the vessel closer together, reducing
residual bleeding and stabilizing the injury site
– Reduces the size of the injured area, making it easier for
fibroblasts, smooth muscle cells, and endothelial cells to
complete repairs.
• Tissue is repaired at the same time as clot retraction.
• Once tissue repair is done, you need to dissolve the clot.
Petechiae are little red dots in skin, from oozing, not enough
platelets, after all the micro tears in your vessels. You are using
your clotting factors right now!
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CLOT DISSOLUTION AND
TISSUE REPAIR: Fibrinolysis
• If a clot stays, it is on the inside of your
blood vessel, so it interferes with the
endothelium from releasing clot factors in
the future. Need to remove the rough clot to
leave a smooth endothelium.
5. Fibrinolysis- Clot dissolution
• Plasminogen = circulating globulin
– Plasmin = THE CLOT BUSTER! proteolytic enzyme, similar to trypsin
– digest fibrin threads, fibrinogen, and other clotting factors
– Significance: removal of tiny little clots
• Plasminogen activators:
– Tissue Plasminogen Activator (TPA), sometimes called the “clot buster”
But is it really?
– Urokinase—found in urine
– streptokinase—from bacteria, so could cause antibody production
– thrombin: now we see negative feedback. It converts plasminogen to
plasmin. If we control thrombin, can control clotting we don’t want. Also need
heparin and antithrombin-3.
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Manipulating Hemostasis- less
clotting
• Important anticoagulants (preventing a clot from forming)
drugs include:
– Heparin—released by basophils and given therapeutically
• High affinity for anti-thrombin III and makes it a more
powerful thrombin inhibitor
• Protamine removes heparin from circulation
• Used in open heart surgery, when blood must pass
through tubing
– Calcium chelators (citrate, oxalate, EDTA, EGTA) used
externally only in collection tubes
Ca++
COO-
COO-
COO– Aspirin
COO• irreversible COX-1 enzyme inhibitor
• This lowers platelet levels of thromboxane, a
prostaglandin needed to make platelets “sticky”
Ca++
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• During heart surgery, you take the blood out of
the patient through a tube, so you line the tubing
with heparin so it does not clot. After surgery,
have to remove heparin from patient. Have to add
protamine (from fish), which competes with
heparin, makes heparin less available to bind to
antithrombin 3, making it less able to bind to
thrombin, so patient does not clot as easily. Add
protamine to a drop of the patient’s blood, see
how long it takes to clot; if too long, add a little
more protamine, to figure out the right dose to
give.
Manipulating Hemostasisless clotting
– Vitamin K deficiency
• Get in green vegs, grains, meats, made by bacteria in large
intestine
• Vitamin K is a fat soluble vitamin needed as coenzyme in
hepatocytes to make factors II, VII, IX and X. Hepatocytes put
these factors into circulating plasma.
• Added issue: Need bile salts to allow fat absorption of Vitamin K;
bile salts are a product of hepatocytes. Need good liver function
to absorb Vitamin K from intestine
• Fat absorption problems--bile duct blockage, prevents uptake of
Vit K
– Coumadin/Coumarin/Warfarin– a natural substance found in certain
grasses
• is a competitive inhibitor of vitamin K activity in hepatocyte;
blocks production of clotting factors II, VII, IX and X.
• These factors must be available in plasma for clotting to occur
• What if patient receiving coumarin eats lots of greens???
– They should decrease their consumption of green veggies.
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Clotting Times: Testing for
deficiencies
•
You do not need to know normal clotting times. It depends on the lot number
of the reagents in your lab.
•
You need to know this:
•
Activated partial thromboplastin time test (aPTT Test)
•
Detects deficiency of Factor VIII
•
Used for heparin treated patients
•
“Prolonged time” = deficiency
•
Prothrombin time test (PT Test)
•
Detects deficiency of Factor VII
•
Used in coumarin patients
•
“Prolonged time” = deficiency
Clotting Times: Testing for
deficiencies
•
For a patient on coumarin, take a ratio of what is normal for them. Their time
divided by normal, should be within a normal range.
•
•
“Warfarin PeT has an APiTite for heparin”
•
PeT= PT time
•
APiTite= APT Intrinsic, heparin
Clotting Times:Testing for deficiencies
Intrinsic
XII
XI
IX
Extrinsic
VII
VIII
Activated partial thromboplastin
time test (aPTT Test)
25-35 seconds
detects deficiency of Factor VIII
Used for heparin treated patients
“Prolonged time” = deficiency
Prothrombin (II)
X
Prothrombin time test (PT Test)
detects deficiency of Factor VII
Used in coumarin patients
“Prolonged time” = deficiency
11-15 seconds
V
Thrombin
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What are those tests used for?


aPTT

Heparin anticoagulant therapy (the target aPTT is usually 1.5 to 2.5 times
that of a "normal" control sample (referred to as the "aPTT Ratio").

Pre-surgery to check coagulation times

Any person with unexplained bleeding

Post-surgery, when adding protamine to remove heparin from patient
PT
often used to check how well anti-coagulant tablets such as warfarin and
phenindione are working.

The drug's effectiveness can be determined by how much it prolongs the
PT (measured in seconds), or increases the INR (a standardized ratio of
the patient's PT versus a normal sample).

Also used to check for a bleeding disorder, liver disease or vitamin K
deficiency, or to ensure clotting ability before surgery.

Most laboratories report PT results that have been adjusted to the
International Normalized Ratio (INR). Patients on anti-coagulant drugs
usually have a target INR of 2.0 to 3.0 (i.e. a prothrombin time 2 to 3
times as long as in a normal patient, using standardized conditions).

Needs standardizing due to tissue factor III having labile biological activity.
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Labtestsonline.org

Manipulating hemostasis to
prevent thrombi & emboli
• A thrombus is formed when
platelets begin to stick to the
wall of an intact blood vessel
– Platelets are often attracted to
plaques – where endothelial and
smooth muscle cells contain lots
of lipids.
– What problems could a growing
thrombus pose?
Above: Normal artery
Below: Same artery with a
large thrombus (arrow)
• If the clot (thrombus) breaks
off and begins to drift in the
bloodstream, it is called an
embolus.
– What problems could an
embolus cause?
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Let’s review some of your endogenous
Anti-coagulants
• Smoothness of the endothelial
lining
• Anti-thrombin III
• Plasminogen
• Fibrin fibers—soak up
thrombin and contain it
• Heparin naturally released by
endothelial cells
• Prostacyclin (a prostaglandin)
promotes vasodilation and is
released by normal, uninjured
endothelial cells
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PATHOLOGY OF PLATELETS
• Thrombocytopenia (Too few platelets)
• Hemophilia
• Disseminated Intravascular Coagulation
Thrombocytopenia
• Idiopathic thrombocytopenia
purpura (ITP)
• Reduced platelet counts:
thrombocytopenia (<100,000/mm3)
tendency to bleed
• Petechiae- small, purple blood
leaks below skin
• Purpura- 1 cm
• Unknown cause, or possibly
autoimmune (seen often after viral
infection) IgA or IgG targets
platelets for destruction. More
common in young and females
• May recover after some weeks,
sometimes not.
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Hemophilia-What symptoms do you suppose
are characteristic of hemophiliacs?
• Refers to several different
hereditary bleeding disorders
with similar signs/symptoms (can
be acquired, too through
autoimmune disorders)
• Hemophilia A
– Results from lack of Factor
VIII. Most common type
(83%)
– X-linked- carrier vs. disease
• Von Willebrand Disease (12p)
– A plasma protein that carries
factor VIII
• Hemophilia B
– Less common. Due to
deficiency of Factor IX
– Also X-linked
Bleeding in the joints (hemarthrosis)
Bleeding and bruising in the soft
tissue and muscles
Bleeding in the mouth from a cut or
bite or loss of a tooth
Nosebleeds for no obvious reason
Blood in the urine (from bleeding in
the kidneys or bladder)
Blood in the stool (from bleeding in
the intestines or stomach)
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• Royalty like to choose particular families to
marry into, or to marry within their family.
Don’t marry your sibling or cousin! You
probably have 4-8 recessive mutations,
bring two parents like that together, kid will
have a problem. Hemophilia is a serious
coagulation disorder, lose a baby tooth and
die from bleeding. The male kids are kept
indoors to protect them from injury, but
become emotional damaged and physically
weak.
Hemophilia
X,X female
X,Y male
= gene for factor VIII is carried on the X chromosome
Y chromosome is short, missing portion
Most common in males, they carry only one allele
• Females
– hemophilic female: what’s her genotype?
– hemophilic “carrier” female: what’s her genotype?
Females can be carriers, and they can get the disease.
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• If father has disease and mother does not, and they have
two boys and two girls, both girls will be carriers; both
boys don’t have the disease.
•
• If the father has no disease and mother is a carrier, one
boy and one girl will not have the disease, and the other
girl will be a carrier, and the other boy will have the
disease.
•
• Females only use one X chromosome, they shut the other
one down, so we get female carriers without the disease.
A calico cat gets its color that way. The colored patches
are the random sites where the father’s color manifests.
Carrier vs. Disease Phenotype
http://www.daviddarling.info/encyclopedia/H/hemophilia.html
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Disseminated Intravascular
Coagulation
– abnormal bleeding and clot formation
– coagulation and clot lysis in
uncontrolled manner—excessive
bleeding and thrombi leading to
ischemia
– due to massive tissue damage (say
from a crushing accident)
– depletion of clotting factors and
hemolysis from damage to red blood
cells in microcirculation from fibrin
fibers
– child birth (HELLP)- hemolysis,
elevated liver enzymes, and low
platelet count. Often misdiagnosed
complication in pregnant women.
http://www.aafp.org/afp/990901ap/829.html
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• DIC is when there is so much tissue
damage that all clotting factors are used,
but there is still bleeding, no clotting factors
are left, and the person bleeds out. HELLP
is DIC during childbirth; the worst
complication of childbirth. HELLP =
Hemolysis elevated liver enzymes and low
platelet count. We can figure that out before
birth if we do a blood test.