White's Hemostasis Lecture Flashcards
Hemostasis refers to
formation of a barrier to blood
Three types of barriers to blood
hemostatic plug, blood clot or thrombus
Primary hemostasis
1) primary hemostasis – primary plug – platelet aggregation
creates a physical plug causing vasoconstriction to reduce blood flow
platelets interact with broken vessel + each other to form primary hemostatic plug
Secondary Hemostasis
fibrin is deposited on the platelets, making the plug even more insoluble
Third stage of hemostasis
fibrinolysis, removal of clot
coagulation factors
soluble blood proteins that form fibrin
Fibrin clot: the goal
involves converting fibriogen into fibrin
fibrinogen is the largest of the plasma protein, constitutes about 4% of blood plasma proteins
another name for circulating inactive coagulation factors
zymogens
these can be activated by proteolysis
what is a zymogen
an inactive protein/enzyme that is activated by site specific proteolysis
In the liver, enzymes with “pre” and “pro” areas ndicate what
pre - for secretion; an area that is cleaved to permit secretion
pro- to keep inactivated; an area that is kept to maintain inactivation
when acted on by the final protein, ________, fibrinogen is converted to fibrin
thrombin
Named factors vs unnamed factors
Factor 1 (F1): fibrinogen F2: prothrombin F3: Tissue factor F4-F8 unnamed F9: Christmas factor F10-F11 unnamed F12 Contact Factor F13 Plasma transglutaminase
What three things happen after a tissue injury to prevent blood loss?
1) vasoconstriction
2) Collagen exposure activates platelets producing a primary hemostatic plug
3) Tissue factors lead to the overlaying of fibrin to produce a fibrin clot.
the “fibrin clot” is composed of fibrin and platelet aggregation
Prothrombin is activated to thrombin by a
serine protease (prothrombinase) serine residues are in the active site then
Thrombin as serine protease
thrombin cuts fibrinogen at ARG-GLY sites
fibrinogen: how it gets activated to fibrin
fibrinogen has 3 domains (D–E–D), 3 subunits (alpha, beta, gamma), and 2 sites for cleaving, A and B (superior/inferior to alpha subunit)
the Alpha subunit can subsequently fit into the gamma site of another fibrinogen’s gamma site: this is the cross linking effect.
alpha (E domain, A site) fits into the gamma (D domain) site, creating a
while cleavage of A peptide uncovers sites in the E domain that are complementary to the D domain
what is the “basic” interaction allowing fibrin to aggregate?
E domains (A sites) interact with D domains on adjacent fibrin (gamma subunit) over and over,
in addition to the A site (E Domain) gamma (D domain) interaction, what other site does thrombin cleave?
the B sites: thenceforth B sites interact to form a 3 dimensional wall
“soft clot”
this is when fibrinogen’s D and E domains interact, and the B sites interact to form the three dimensional wall
E–D domain interactions use ___ bonds in _______ formation
H bonds in soft clot formation
Hard Clot Formation
Use covalent bonds between NH2 of glutamine and NH3 of lysin
What bonds form the hard clot?
NH2 of glutamine and NH3 of lysine, catalyzed by transglutaminase (Factor XIIIa)
FXIII —> FXIIIa via
thrombin, which activates transglutaminase
Prothrombin’s post-translational modification is
important for clot localization
How is protrhombin modified?
a glutamic acids gets an additional carboxyl group that adds a -2 charge to an R group on amino acid = gamma carboxyglutamate.
What is protrhombin’s post-translational modification called
gamma-carboxyglutamate
glutamic acid on prothrombin receives a corboxylic acid group that has a -2 charge
Why is the additional charged group added to glutamic acid on prothrombin important?
Damaged tissue rexposes calcium (+2 charge) at the site of injury, so carboxyglutamine can bind it perfectly with the -2 charge added.
in turn Calcium binds platelets which have exploded uncovering negatively charged phospholipids as they invert
Platelet phospholipids
invert after binding to exposed collagen
negative charge on inverted phospholipids binds calcium which is also stabilized by the carboxyglutamate in the thrombin
Vitamin K
caryboxylation of prothrombin requires vK
4 enzyme involved in carboxylation of glutamic acid in prothrombin
Vitamin K Hydroquinone–> oxidized to Vitamin K epoxide by Carboxylase (activated by VKH)–> reduced to VKH by Epoxide Reductase
warfarin and Dicoumerol: competitive inhibitors of epoxide reductase.
Coumadin
warfarin, competitive inhibitor of Epoxide Reductase: prevents Vk from activating enzyme responsible for carboxylating glutamic acid in prothrombin
Dicoumerol
competitive inhibitor of epoxide reductase
warfarin facts
active ingredient in rat poison, patients have to be watched carefully
WARF = “Wisconsin Alumni Research Fund” patented by university of wisconsin.
Clotting cascade of _______
serine proteases, contain serine residue in active site of proteases
exist as zymogens- inactive enzymes made by proteolytic cleavage
Intrinsic PW
workhorse of the clotting cascade, does not requires a protein outside of the blood to be activated
Extrinsic PW
the “SPARK” to get the clotting cascade going
Intrinsic PW factors
12, 11, 9, and 8:, all activated. Importantly,
(F8/F9)a
- Intrinsic PW begins with ______ being activated by ______
contact factor (12) being activated by active form of pre-kalikrein (Kalikrein), a protease
XII (surface activation) —> XIIa
- Intrinsic PW’s second step is activation of ______ by —-
XI by Calcium-bound XIIa
- Intrinsic PW’s third step is activation of ____ by ____
IX by XIa+PF3+Calcium—> IXa
PF3 is the inverted phospholipid on the platelet that binds calcium
- Intrinsic PWs fourth step is the formation of
IXa - VIIIa complex
Thrombin activates VIII
Start of Common PW begins with
(IX–VIII) = Tenase
Tenase activate X —> Xa in the presence of PF3 and Calcium
Xa combines with FVa to form PROTHROMBINASE
V is activated by thrombin
Which factor is common to intrinsic and extrinsic?
X
what two factors are activated during the intrinsic PW
VIII and V
Prothrombinase
Va+Xa
Xa–Va activate
This is the prothrombinase that activates thrombin
Hemophilia is deficiency in what factors
Type A is VIII deficiency and Type B is IX deficiency
Amplification Cascade
a handful of XII causes a massive clotting cascade signal, each factor is increased at each stop of the clotting cascade
Extrinsic Pathway
Activated by a component not normally in the blood, factor III (tissue factor)
Tissue factor activates VII
How EX PW begins
factor III activates factor VII
VIIa combine with another factor III to form a complex
VIIa-TF complex
The rest is the common PW
Vlla+TF complex activate X—Xa while thrombin activates V to Va
Va+Xa form Prothrombinase
Prothrombinase activates prothrombin –> thrombin
thrombin continues cascade
Common PW is just
Factor X to the fibrin clot formation
How is thrombin active if thrombin is actively produced?
~ 1% of FVII circulates as FVIIa, but does not contact damaged tissue
After damage, VII and XIII tissue factor combine to form complex that activates FX
Platelets release FVa, released from platelets
forms FXa–FVa complex (prothrombinase comlex) which gets things going
Why is Extrinsic important outside of its own contributions to the clotting cascade? How does it relate to the intrinsic PW?
It sets the intrinsic in motion by setting VII into motion (VIIa)
Vitamin K Dependent Factors: 3 factors that aren’t glutamic acid that require vitamin K activated carboxylation
X, IX, VII
Positive Feedback Mechanism for clotting factors
Thrombin, XIa, Xa, VIIa
Partial THromboplastin Time
PTT
Clotting time from Factor XII to fibrin clot –» intrinsic pathway, reference is 35 sec
Prothrombine Time
PT
Clotting time from factor VII to fibrin clot —> extrinsic and common pathwayas
reference time = 10 - 12 sec
Clotting in vivo requires a
TF: tissue factor, initiating component (FIII)
So what is REALLY be tested for in vivo?
VIIa-TF complex, which activates FIX
FIX combines with activated VIII to activate FX, which combines with thrombin-activated V
to activate prothrombin –> thrombin
IXa –VIII (Tenase)
Regulation I: How fibrin is turned off
thrombin binds it and inactivates it
Thrombin can cut FVIIIA to inactivate form FVIIIi
Thrombin can cut Xa to Xi
Thrombin can cut Va to Vi
hrombin can cut prothrombin to create an inactive form
Regulation II: How Thrombin works to this end
combines with a protein called Thrombomodulin to create TTM complex
this cleaves a protein called Protein C into active protein Ca
Protein Ca and others inactive FVa and FVIIIa which decreases thrombin
Anticlotters: preventing clotting
warfarin and dicoumerol
Protects platelet integrity
aspirin
Heparin
activates antithrombin III by binding to thrombin and Xa, which it inactivates
TPA
tissue plasminogen factor
percursor to plasmin, dissolves clot
fibrinolysis
first genetically engineered human protein to be approved for use in humans, given to remove clot, aslo after stroke if given early
