Intro to Hemostasis II

Question 1

Antithrombin

Answer 1

• serpin
• targets serin proteases (most importantly Xa and thrombin)
• inactivates the target by binding an arginyl residue in its reactive site loop to the serine in the catalytic site of the protease
• both serpin and protease are inactivated and cleared

Question 2

Heparin

Answer 2

• key cofactor for antithrombin - accelerating its rate of protease inactivation
  1. shorter form of heparin. causes an allosteric conformational change in antithrombin that allows more efficient binding to and inhibition of the target protease (inact Xa but not thrombin)
  2. longer form. binds to antithrombin + serine protease target to bring closer in proximity (need for thrombin inactivation)

Question 3

What is protein C?

Answer 3

• vitamin K-dependent serine protease
• zymogen in circulation
• short T1/2 (8-10 hrs)

Question 4

Protein C activation

Answer 4

When thrombin is generated from prothrombin, it binds to thrombomodulin, a transmembrane protein constitutively expressed on endothelial cells. Once bound to thrombomodulin, thrombin’s procoagulant activity is neutralized, but the thrombin-thrombomodulin complex on the cell surface can bind to and activate protein C (APC)

Question 5

Protein C-protein synthesis regulation of coagulation

Answer 5

APC cleaves/inactivates cofactors Va and VIIIa -> decreased thrombin
APC + S -> change in structure of APC and moves its active site closer to the membrane surface

Question 6

Protein C deficiency ->

Answer 6

increased thrombotic risk

Question 7

Explain what factor V Leiden is

Answer 7

• AKA APC resistance
• A point mutation leads to a change at aa 506 of the protein from glut -> arg
• Va becomes resistant to protein C and remains activated longer than it should

Question 8

Describe how tissue factor pathway inhibitor (TFPI) functions in regulating coagulation

Answer 8

• expressed constitutively by endothelial cells
• inhibitor of the extrinsic pathway
• TFPI binds to Xa’s active site as it’s produced by the extrinsic tenase complex (VIIa+TF), inactivating the tenase -> complex of Xa+TFPI+VIIa+TF
• same idea with XIa but with less affinity

Question 9

Fibrinolysis is

Answer 9

the process of clot breakdown that occurs following clot formation, allowing eventual repair of the damaged blood vessel following injury. It begins as soon as the clot begins forming

Question 10

Role of plasmin in fibrinolysis:

Answer 10

key enzyme which is a serine protease cleaved from its zymogen. It can cleave fibrinogen and fibrin -> fragments (fibrin degradation products/FDP) which are detected in the blood. It also breaks down ECM proteins to help remodel/repair the damaged vessel

Question 11

How plasminogen is activated

Answer 11

by t-PA or U-PA (as well as minimally by contact factors of the intrinsic pathway)

t-PA: efficiently activates plasminogen to plasmin upon binding to fibrin, and as a clot forms, t-PA and plasminogen bind to the fibrin being generated, localizing plasmin generation to the site of the clot

u-PA: prourokinase becomes bound to the clot and is then cleaved, primarily by already generated plasmin, into its active form which can then contribute to further clot lysis

Question 12

Plasminogen activation inhibitor 1 (PAI-1)

Answer 12

serpin that binds to and inactivates t-PA (and u-PA?)

Question 13

Alpha2-antiplasmin

Answer 13

serpin that is the primary plasmin inhibitor in the blood. It binds to and inactivates plasmin (1:1), preventing systemic fibrinogen degradation

Question 14

Thrombin-activatable fibrinolysis inhibitor (TAFI)

Answer 14

zymogen that circulates in the blood in complex with plasminogen. Once activated, it is an exopeptidase that removes amino acids from the c-terminal of proteins -> less plasminogen binding sites on fibrin -> decreases available plasminogen to t-PA and u-PA

Question 15

List some of the mechanisms the endothelial cell lining uses to prevent clot formation in the resting state

Answer 15

Anticoag:

1. Expression of heparin sulfate and dermatan sulfate, which act as cofactors for antithrombin and heparin cofactor II, respectively.
2. Expression of thrombomodulin, which acts as a cofactor with thrombin for activation of protein C.
3. Expression of TFPI to inhibit the extrinsic Xase complex.

Antiplatelet:

1. Synthesis and secretion of prostacyclin (PGI2) and nitric oxide (NO), which prevent adhesion of activated platelets and cause vasodilation.
2. Expression of an enzyme that rapidly metabolizes ADP (a potent platelet agonist) to AMP and adenosine (a potent inhibitor of platelet function)

Question 16

Primary hemostasis:

Answer 16

platelet adhesion, aggregation, and activation

• VWF binds to subendothelial matrix -> adherence of platelets
• platelets change shape, are activated and aggregate -> release of platelet granules
• phosphatidylserine is translocated from the inner leaflet to the outer leaflet

Question 17

Secondary hemostasis:

Answer 17

activation of the coagulation cascade and formation of the fibrin clot

• TF is exposed to VIIa -> initiation of the extrinsic pathway
• act IV and X
• Xa + Va + Ca2+ -> act prothrombin -> thrombin, which act XI, VIII and V -> amp
• propagation phase -> thrombin burst -> fibrinogen -> fibrin
• fibrin network around activated platelets, thrombin act XIII -> cross-links -> stabilized clot