Hemostasis and Coagulation Cascade

Question 1

Zymogen and Serine Protease

Answer 1

Zymogen, aka proenzyme – inactive enzyme precursor

Serine protease: an enzyme that cleaves peptide bonds
Serine is the nucleophilic amino acid in the enzyme’s active site
Cleavage of disulfide bonds causes activation

Question 2

Extrinsic 10ase

Answer 2

TF (VIIa), factor 10 (inactive), Ca2+, and phosphotidylserine to cleave the factor 10 and make it active

Question 3

Gamma - Carboxyglutamate (GLA)

Answer 3

aka Gla residue
Post-translational modification of glutamate
Found in Prothrombin, Factors VII, IX, and X, and proteins C, S and Z

Binds calcium ions:
Aids binding of coagulation cascade proteins to membrane components
Necessary for cleavage activity of the serine proteases

GLA = binds Ca2+ to allow molecules to bind to membrane surfaces
Factor 7 in extrinsic
9- intrinsic pathway
10 – cleaves thrombin

Proteins C, S, and Z – involved in anti-coagulations

Question 4

Synthesis of GLA

Answer 4

Requires vitamin K
Oxidation forms reactive carbanion and vitamin K epoxide
Non-enzymatic carboxylation forms γ-carboxyglutamate
Epoxide is reduced to quinone and then hydroquinone
Warfarin prevents recycling of vitamin K

Excess vitamin K can overcome the inhibition because second reductase is insensitive to Warfarin

Question 5

Activation of Protein C Pathway

Answer 5

Thrombin is inactivated by thrombomodulin on endothelial surface and also complex activates protein C and combines with protein S and inactivates factors 5 and 8
Inhibits coagulation cascade

Question 6

Activation of Antithrombin

Answer 6

Antithrombin is serine protease inhibitor; inactivates any of the factors and inactivation activity is accelerated by cell surface heparin – derived from heparin and can stimulate antithrombin (inactivation of thrombin and factors)
Inhibits coagulation cascade

Question 7

Factor Z and Z Related Protease Inhibitor

Answer 7

Factor Z and Z related protease inhibitor – inhibits factor XIa
Inhibits coagulation cascade

Question 8

Fibrinolysis

Answer 8

Resolve fibrin clots via plasmin
Degrades fibrin and pulls apart clots
T-PA activated
Lysine residues bound to plasminogen to keep it in clot so readily available and cleave fibrin
Urokinase: ECM degradation and fibrin clot degradation

Question 9

Regulation of Fibrinolysis: Prevention

Answer 9

Primary defense: alpha2 antiplasmin circulation in bloodstream to act on plasmin to inhibit it

Thrombin-activatable fibrinolysis inhibitor
(TAFIa) – acts negatively on plasmin so stops dissolution of the clots; is a carboxypeptidase; prevents plasmin from binding to fibrin

Plasminogen activator inhibitor: negatively affects t-PA so plasminogen cannot be converted to plasmin

Question 10

Thrombin: Coagulation Action

Answer 10

Cleavage of fibrinogen to form fibrin
Activation of factor XIII (to crosslink fibrin)
Activation of factors V and VIII (Cofactors for factor Xa and factor IXa, respectively)
Activation of factor XI

Question 11

Thrombin: Anti-Coagulation Action

Answer 11

Activation of protein C

Requires binding to thrombomodulin

Question 12

Thrombin: Fibrinolysis Inhibition

Answer 12

Activation of thrombin-activatable fibrinolysis inhibitor (TAFI)
Requires binding to thrombomodulin

Question 13

NO

Answer 13

Nitric Oxide (NO): inhibits platelet adhesion and aggregation by elevating levels of cGMP

Question 14

Thrombomodulin

Answer 14

Thrombomodulin: glycoprotein that binds thrombin, which then cleaves protein C to yield activated protein C; this is in combination with protein S degrades factors Va and VIIIa, limiting their actions

Question 15

t-PA

Answer 15

t-PA: tissue plasminogen activator; activates plasminogen to plasmin, which digests fibrin; the action of t-PA is opposed by plasminogen activator inhibitor-1 (PAI-1)

Question 16

IX Activation

Answer 16

Intrinsic pathway via XIa

Extrinsic pathway via VIIa

Question 17

Endogenous Anticoagulants

Answer 17

1. Antithrombin III: inhibits IXa, Xa, XIa, XIIa
2. Protein C or S: vitamin K dependent; inactivates Va and VIIIa
3. TFPI: produced via endothelium; inactivates VIIa, therefore inhibiting IX and X

Question 18

VIIa Activation

Answer 18

activates Xa and IXa

Question 19

IX Activation

Answer 19

IX activates X

Question 20

PT

Answer 20

Extrinsic Pathway (initiating pathway)
2, 5, 7, 10, and fibrinogen

Question 21

PTT

Answer 21

Intrinsic Pathway (amplifying pathway)
2, 5, 8, 9, 10, 11, 12, and fibrinogen

Question 22

GPCR

Answer 22

G protein coupled receptors
Ligands bind to cause conformation change to make GTP from GDP in order to activate and dissociate complex
alpha is a GTPase to make GTP to GDP and inactivates everything again
2 regulatory pathways affected: PLC and adenylyl cyclase

Question 23

Platelet Activation: Adhesion

Answer 23

Exposed collagen and subendothelial vWF
Collagen binds to GP Ia-IIa and GPVI
vWF binds to GP Ib-IX-V
Thrombin binds GP Ib-IX-V, PAR-1, PAR-4

Collagen and thombin activate phospholipase to cleave PIP2 to form 2 second messengers:

1. DAG: activates protein C to cause platelet responses and release granules
2. IP3: stimulates Ca2+ release to cause platelet responses (affects shape)

Question 24

Release of Platelet Factors

Answer 24

1. Serotonin
2. TxA2: Ca2+ stimulates phospholipase to cleave arachidonic acid to form TxA2 and activate other platelets
3. ADP: released by DAG to stimulate platelets

Question 25

Platelet Aggregation

Answer 25

GP IIb-IIIa binds fibrinogen to bring platelets together to make primary platelet plug Fibrinogen cleaved to fibrin via thrombin to make secondary stronger plug

Question 26

Platelet Inactivation

Answer 26

Prostacyclin inhibits platelet activation and released by endothelial cells Acts through GPCR to increase cAMP Aspirin inhibits COX-1 to prevent TxA2

Question 27

Fibrinogen Structure

Answer 27

N terminals are held together at the center by fibrinopeptides with a high negative charge bound together by disulfide bonds When activated, negative charge decreases so platelets can interact and fibrin can build Factor XIIIa (activated by thrombin) is a transglutaminase and cross links fibrin molecules via covalent bonds