Biochemistry of clotting

Question 1

Plasma

Answer 1

Blood + anti-coagulant, then spun down

Clotting factors still in zymogen form

Question 2

Serum

Answer 2

Blood is allowed to clot, then spun down

Clotting factors inactivated, fibrinogen depleted

Question 3

Platelets

Answer 3

formed from megakaryocytes (WBCs synthesized in marrow)
non-nucleated
2-3 microm in diameter
half life: 5-9 days
150-400 x 10^9/L
activated by thrombin or ADP

Question 4

Physiological events in thrombosis

Answer 4

Endothelial damage
platelet adhesion
platelet aggregation
Fibrin clot formation

Question 5

Overview of clotting cascade

Answer 5

Initiator (tissue damage)
activation of clotting factors
Thrombin catalyzes fibrinogen (soluble) –> fibrin (insoluble)

Question 6

Extrinsic pathway

Answer 6

Initiation
components are platelet-membrane bound

Vascular injury
Tissue factor
Factor VII- tissue factor --> Factor VIIa-tissue factor
VIIa catalyzes Factor IX --> Factor IXa
VIIa catalyzes Factor X --> Factor Xa

Question 7

Common pathway

Answer 7

Factor Xa catalyzes prothrombin –> thrombin
Thrombin catalyzes V –> Va, which catalyzes above
Thrombin catalzes fibrinogen –> fibrin
Fibrin + Factor XIIIa (converted by thrombin) –> cross-linked fibrin

Question 8

Intrinsic pathway

Answer 8

Propagation

Factor XI –> XIa
XIa catalyzes IX –> IXa
IXa catalyzes X –> Xa

Thrombin catalyzes XI –> Xia, VIII –> VIIIa

Question 9

Fibrinogen

Answer 9

6 polypeptides : a2b2gamma2
shaped like a dumbell
Three globular domains linked by triple helixes

Question 10

Fibrin formation

Answer 10

Fibrinogen is cleaved at 4 sites (N-termini)

Fibrinopeptides A&B removed, leaving behind alpha and beta knobs

Question 11

Fibrin polymerization

Answer 11

New N-termini on activated fibrin can bind terminal domains on other fibrins
Dimer still soluble
polymer insoluble

Question 12

Fibrin cross-linking

Answer 12

Introduced by XIIIa
Covalent bonds between adjacent terminal domains
XIIIa also crosslinks fibrin clot to surrounding tissue

Question 13

Role of vitamin K in clotting

Answer 13

Vitamin-K dependent carboxylation required for biological activation
Normally, gut flora synthesis sufficient
Vitamin K injection in newborns
Glu –> Gla

Question 14

Gla protein - role in clotting

Answer 14

Gla binds Ca, conformation change
Ca-Gla-proteins can bind phospholid membranes (supplied by activated platelets)
Ensures that fibrin formation occurs at the site of injury instead of flowing in blood
Gla required for membrane binding (need vit K) of VII, X, IX, prothrombin ( = vit K proteins)

allow concentration, orientation, localization

Question 15

Factor XII

Answer 15

Deficient patients don’t bleed
Probably has non-coagulant functions
Minor role in hemostasis compared to TF-fVIIa-TFPI complex
Activated by poly-phosphate on the activated platelet surface
Resulting XIIa activates XI –> XIa, generating thrombin for further platelet activation after TF-fVIIa-TFPI complex is formed
XII deficiency protects against arterial thrombus formation

Question 16

Termination of clotting

Answer 16

dilution
trapping of proteases in fibrin clot
serine protease inhibitors (Serpins) such as antithrombin bind to active sites of thrombin and forms inactive complex
Antithrombin deficiency: risk of thrombosis
Heparin accelerates antithrombin-thrombin reaction (anticoagulant)

Question 17

Thrombomodulin - Protein C - Protein S pathway

Answer 17

Thrombomodulin appears on endothelial cell membrane proteins
converts thrombin from a procoagulant to an anticoagulant
which then activates Protein C (protease) with protein S
leads to inactivation of Va, VIIIa

Question 18

Plasminogen activation

Answer 18

Activator (protease - tPA, uPA, streptokinase/staphylokinase)
converts plasminogen –> plasmin (enzyme)

Question 19

Role of plasmin

Answer 19

cleaves triple helix of the fibrin polymer

forms D-dimers, which are soluble and cleared by the liver

Question 20

Lab coagulation tests

Answer 20

Prothrombin time - INR

Activated partial thromboplastin time (APTT) - also PTT

Question 21

Prothrombin time

Answer 21

Plasma incubated with thromboplastin (phospholipid + tissue factor), then Ca added
Measure clotting time of extrinsic & common pathway
10-13 s but varies

Question 22

INR

Answer 22

Prothrombin time (in seconds), varies among labs
Differences in batches of tissue factor

INR = [patient time/reference time ]^ISI
ISI = international sensitivity index 

Normal people, INR = 1
anticoagulant drugs >1

Question 23

Activated partial thromboplastin time

Answer 23

Plasma + kaolin + partial thromboplastin (phospholipid, no tissue factor) , add Ca
Measure clotting time (intrinsic/common pathways)
24-37s, varies