Exam 3: Coagulation and Dissolution of a Blood Clot

Question 1

Overview of coagulation: 3 steps & “3-ish big items that are involved”

(putting into words the initial picture from ppt lecture)

Answer 1

• damaged blood vessel - releases clotting factors
• clotting factors turn prothrombin to thrombin
• thrombin turns fibinogen (soluble) to fibrin (insoluble) - this will go to step 3

1. damaged blood vessel - release of clotting factors
2. plt plug + vasoconstriction to site (limits blood flow)
3. development of clot - fibrin strands adhere to the plt plug to form an insoluble clot

Question 2

Normal Hemostasis: describe the 3 pathways

Answer 2

• a balance between generation of hemostatic clots and uncontrolled thrombus formation
• Extrinsic pathway: plasma-mediated, initiation of hemostasis
  
  • AKA primary hemostasis
  • key: TF (tissue factor)
• Intrinsic pathway: amplifies and propagates hemostasis
  
  • AKA secondary hemostasis
  • key: thrombin
  • ppl with congenital issues can still clot, but they might get into trouble in “dire, stressful situations”
• Common pathway: results in an insoluble fibrin clot

“Some extrinsic force causes you to bleed, then your intrinsic forces must stop the bleeding”

Which one is predominant in hemostasis? In general, we are anticoagulated!

Question 3

Preop Coag testing

Answer 3

• should be based on pt’s history and planned surgery
  
  • BOARDS QUESTION
• balance between risk of surgical bldg and risk of developing postop thrombembolism
• agents
  
  • antiplatelets: ASA
  • anticoagulants: coumadin, Heparin
  • thrombolytics: TPA
  • procoagulants: FFP
• try to remember the test that goes along with each drug (ex: warfarin will be INR to test levels)

Question 4

Mechanism of Blood Coagulation

Answer 4

• hemostasis is precisely regulated by interactions between blood vessel walls, circulating plts, and clotting proteins in the plasma
• fibrinolysis is the orderly breakdown of a stable blood clot
• > 50 substances that affect blood coagulation have been identified
  
  • fibrin disc in 1800s
• normally, anticoagulants predominate but when a vessel is ruptured, procoagulants are activated

Question 5

normal hemostasis steps (4)

Answer 5

• vasoconstriction
• plt plug
• clot formation
• clot dissolution

Question 6

what is down under the endothelium

Answer 6

TF!

• when there’s a break, tissue factor and thrombin interact
• they signal that something is wrong, so plts start coming
• subendothelial TF initiates plasma-mediated hemostasis via the EXTRINSIC pathway
• the intrinsic pathway further amplifies thrombin and fibrin generation
• plts adhere to exposed collagen to undergo activation, resulting in recruitment and aggregation of additional plts

Question 7

what is visoelastic testing

Answer 7

TEGS and ROTEMS

• Piston inside of a cup of blood. In one of them the cup moves and in one of them the piston moves.
• But basically spins and tells you how long it takes for a clot to:
  1. form
  2. become stable
  3. dissolve

but the results take 30-45 minutes

Question 8

what categories do each of these belong to:

plts

coagulation factors

Answer 8

• cells = RBCs, WBCs, plts
  
  • plasma = water, proteins, globulins
    
    • globulins = alpha, beta (coagulation factors), gamma (antibodies)

Question 9

step 1: vasoconstriction

Answer 9

• normally, vasc endothelium provides a nonthrombogenic surface
  
  • “anti-plt, anticoagulant, pro-fibrinolytic” = all just mean NO CLOT
• Damage to the endothelium exposes the underlying extracellular matric and elicits a contraction (vasoconstriction)
  
  • thrombin, hypoxia and high fluid sheer stress can also induce prothrombotic endothelial changes
  • “high fluid sheer stress” = stress over an atherosclerotic plaque, or pulmonary stenosis

“damage → reflex vasoconstriction”

Question 10

step 2: formation of a plt plug

Answer 10

• when plts are exposed to the extracellular matrix in damaged endothelium, they undergo a series of biochemical and physical alterations (grow arms!)
• 3 major phases
  
  • adhesion - sticky plts!
  • activation
  • aggregation

Question 11

tell me about plts

(nml concentration, life span of a plt, etc)

Answer 11

• plt = thrombocyte
  
  • thrombus/clot + cells
• formed in bone marrow
• nml concentration is 150,000-400,000 per microliter
  
  • spontaneous bldg if <50,000 u/L
  • lethal if <10,000 u/L
• life of a plt is 8-12 days
  
  • preop interview! they should have held antiplts for 8-12 days!

Question 12

name the 5 receptors on plts

Answer 12

• ADP: P2Y12
• TXA2: TP
• collagen: GP1a, GPVI
• vW factor: GP Ib-IX-V
• fibrinogen/ vWF

Question 13

plt adhesion

Answer 13

• going from lazy river → sticky, with feet
• conformational change which enables plts to stick and not be washed away
• exposure to subendothelial matrix proteins allows plts to undergo a conformational change to adhere to the vasc wall

Question 14

von Willebrand Factor (vWF)

Answer 14

• vWF is produced in the endothelium and plts. it is released by endothelial cells and activated by plts
• vWF’s primary fn is to bind other protiens
• it’s important as a bridging molecule between the subendothelial matrix and plts, forming cross links
  
  • glycoprotein IIb/IIIa = plt-plt
    
    • ​reopro, aggrastat, integrilin
  • glycoprotein Ib/factor IX/factor V receptor complex = plt-vessel
  • (these are the names of the receptors)
• ​primary thing that enables binding to wall or other plts

Question 15

enrichment: GPIb-V-IX complex binds vWF, causing what?

Answer 15

allows plt adhesion (to each other and to the vessel wall) and plt plug formation at sites of vasc injury

absence of the GP1b-5-9 receptor is known as Bernard-Soulier syndrome

Question 16

vWD - von Willebrand Disease

how it’s activated

incidence

where it shows up

Answer 16

• vWF is mainly activated in conditions of high blood flow and shear stress
• 1/100 ppl, but clinically significant cases 1/10,000
• defic of vWF thereshow show primarily in organs with small vessels, such as skin, GI and uterus
  
  • dx’d by measuring amt of vWF in a vWF antigen assay and the functionaliyt of vWF with binding assays
  • factor VIII (factor 8) is also measured (they’re buddies)
• think of woman who has really heavy menstrual bldg, nose bleeds, gums bld easily, frequent bruising*
• • could show up in post-partum bldg*

Question 17

Types of vWD (3 + pseudo)

Answer 17

• Type 1 vWD (60-80%) - failure to secrete vWF into circulation or vWF being cleared more quickly than normal
  
  • mild, often undx’d until bldg following surgery, easy bruising or menorrhagia (abn heavy periods)
  • drug that helps them secrete the vWF: DDAVP/desmopressin
• Type 2 vWD (15-30%) - qualitative defect and bldg varies. Has 4 subtypes.
  
  • ↓ ability to bind GP1b
  • ↓ ability to bind VIII
• Type 3 vWD - most severe, homozygous defective gene, NO production of vWF
  
  • ​leads to extremely low levels of VIII, since it does normally protects FVIII from proteolytic degradation
  • (NIH - vWF is a carrier protein for FVIII)
  • can’t give them DDAVP - they need exogenous vWF
  • “Type 3, I have no vWF in me”
• Platelet type - aka pseudo-vWD - this is a defect in the plt’s GP1b receptor
  
  • not technically an issue with vWF
  • the plt GP1b is abnormal

We need to know what type of vWD they have

“von Willebrand has EIGHT close mates” - vWF and Factor VIII are best buds

Question 18

What meds do we use that are GPIIb/IIIa inhibitors? (3)

And what class are they?

What’s their MOA?

Answer 18

• antiplts!
• Reopro = abciximab
• Integrilin = eptifibatide
• Aggrestat = tirofiban
• Blocks the ability of fibrinogen to form around aggregated plts
  
  • as a result, no fibrinogen bridging of plts to other plts can occur
• integrilin and aggrestat T1/2 2-4 hrs, so usually in gtt form
• there’s no antidote, so bldg could occur elsewhere in the body and you’d have to wait it out or give plts
• ex of dr. funk’s when they’d be on the gtts in the ICU but they’d be bldg from IV sites or ETTs

Question 19

plt activation

Answer 19

• after plts adhere to the damaged endothelial wall, several intracellular signaling pathways are activated when ligands bind to plts receptors and a series of physical and biochemical changes occur
• also pls dev pseudopod-like membrane extensions (FINGERS AND FEET) to ↑ plt surface area

Question 20

plt recruitment

Answer 20

plts rel granular contents, resulting in recruitment and activation of additional plts

• some of the other contents
  
  • ADP, ATP
  • serotonin, histamine
  • Ca++
  • vWF, FV, VII, XI, XIII
  • fibrinogen
  • PAI-1, TFPI
  • thrombospondin, fibronectin

Question 21

IIb/IIIa inhibitors:

what makes abciximab (ReoPro) different from eptifibatide (Integrilin) and tirofiban (Aggrastat)

Answer 21

• it binds irreversibly to plts
• time to restore plt fn is 24 hrs, compared to 4-6 hrs for integrilin and aggrastat

Question 22

thromboxane A2 inhibitors

Answer 22

• Aspirin – inhibits the ability of COX enzyme to synthesize the precursors of thromboxane within platelets
• Naproxen – nonselective COX inhibitor

Question 23

ADP and plts

Answer 23

• P2Y12 receptors further amplify the response to ADP and draw forth the completion of aggregation

Question 24

ADP receptor antagonist meds?

(P2Y12)

prodrugs and direct-acting ones

Answer 24

• prodrugs (have to go thru the liver to start working)
  
  • ticlopidine = Ticlid
  • clopidogrel = Plavix
  • prasugrel = Efient
• direct-acting (more effective)
  
  • ticagrelor = Brilinta
  • cangrelor = Kengrexal
• before ADP inhbitiros, the only antiplt on the market was ASA
• researchers began in the 1970’s to look for drugs to target other important signaling pathways
• remember that 15% of pts who metabolize plts differently

Question 25

plt aggregation

which receptor links what

Answer 25

• plt aggregation completes the formation of a plt plug
• activators released during the activation phase recruit and amplify the response of additional plts to the site of injury
• newly activated glycoprotein IIb/IIIa receptors on the plt surface bind fibrinogen to provide cross-linking with adjacent plts
  
  • covalent bond = strong, tight

image of 2 plts stuck together:

• GP IIb-IIIa = vWF + fibrinogen “net” (plt to fibrinogen)
• GP Ib-IX-V (1b-9-5) = vWF + other vWF (plt to each other)

Question 26

step 3: formation of blood clot

Answer 26

• Following platelet adhesion, activation and aggregation…the blood clot begins to form
  
  • The plasma protein fibrinogen breaks down to produce fibrin, which subsequently becomes cross-linked into a stable mesh
• Coagulation factors are then activated and initiate the coagulation cascade
• Soluble fibrinogen is converted to insoluble fibrin
• The key step in blood clotting is the conversion of fibrinogen (I) to fibrin (Ia) by thrombin (IIa)
• the body is already incorporating plasminogen into the clot
• thrombin = makes clot strong
  
  • “strong-bin”

Question 27

• alpha angle on TEG/ROTEM
• MCF - maximum clot firmness

Answer 27

• alpha - leading up to how strong of a clot it’s going to get
• MCF - how strong the clot it

Question 28

coag cascade summary

Answer 28

• The classic description of coagulation includes initiation of this chain reaction via either the intrinsic or extrinsic pathway
  
  • Intrinsic pathway is triggered when blood contacts a negatively charged surface (i.e. exposed subendothelial collagen)
  • Extrinsic pathway is activated when blood contacts cells outside the vascular endothelium.
    
    • Nonvascular cells express a membrane protein called tissue factor (III) which initiates this pathway
  • Common Final Pathway is where the intrinsic and extrinsic pathways converge with the activation of factor X

Question 29

coagulation factors:

how are they named?

where are they synthesized?

which ones are Vit-K dependent for utilization?

Answer 29

• Were identified using Roman numerals assigned in order of discovery
• Most coagulation factors are enzymes with some exceptions
  
  • vWF and Tissue Factor (III) are glycoproteins
• Most are synthesized in the liver with some exceptions
  
  • Calcium (IV) comes from diet
  • Von Willebrand is synthesized in endothelial cells and platelets
• Factors II, VII, IX and X are Vitamin K dependent for utilization (coumadin) **Test question for sure!
• Circulate in an inactive state
• A lower-case letter “a” is the active enzyme
  
  • E.g.: Inactive prothrombin is referred to as factor II and active thrombin is identified as factor IIa

Question 30

intrinsic pathway of coagulation

Answer 30

• “Contact Activation System”
• Begins with damage to the blood vessels themselves
• Formation of the primary complex on collagen and thrombin generation by way of factor XII and ultimately merges to the common pathway an activates factor X
• The formation of this primary contact in initial clot formation may prove to be a minor role as patients with these severe factor deficiencies do not have bleeding disorders
• May be more involved with inflammation, amplification, and propagation of the hemostatic response to maximize thrombin generation

Question 31

***TEST***

Which factors are Vitamin-K dependent for utilization?

Answer 31

2, 7, 9, 10

Question 32

Interchangeable names for :

Factor I

Factor II

Factor III

Factor IV

Answer 32

Factor I = Fibrinogen

Factor II = Prothrombin

Factor III = Tissue Factor

Factor IV = Calcium

Calcium is always 4! Ca-Ch blockers is class IV, and now this!

Question 33

Extrinsic pathway of coagulation

Answer 33

• “Tissue Factor Pathway”
• Initial step in plasma-mediated hemostasis
• Following damage to the blood vessel, Factor VII comes into contact with tissue factor (which is prevalent in the sub-endothelial tissues surrounding vasculature) and forms an activated TF-VIIa complex
• The TF-VIIa circulating the plasma activates factor X to promote the conversion of X to Xa.

7-(W)TF-Extrinsic - “7 ways to extrinsic-ally cause bleeding, wtf”

For the test, we don’t have to memorize all of this, but we DO need to know where a drug works (ex: heparin works in the coagulation cascade, has nothing to do with plts)

Question 34

Common pathway of Coagulation

Answer 34

• Common to both extrinsic and intrinsic
• It depicts thrombin generation and subsequent fibrin formation - prothrombin (II) is cleaved by activated factor X to produce thrombin (IIa)
• “10 cleaves 2 (prothrombin) into 2a (thrombin)”
• Signal amplification
• Thrombin activity
  
  • Fibrinogen (I) to fibrin
  • activates platelets and factor XIII (fibrin-stabilizing factor)
  • converts inactive cofactors V (proaccelerin) and VIII (antihemophilic factor A) to active forms
  • activates factor XI (plasma thromboplastin antecedent) and up-regulates tissue factor
  • stimulates vascular endothelium to express downregulation of fibrinolytic activity

Question 35

Ppl with Factor 13 deficiency

Answer 35

can make clots but they won’t stay

Question 36

Blood clot

(somewhat repetitive but it had its own slide for reinforcement)

Answer 36

• Prothrombin gets activated to thrombin
• Thrombin activates fibrinogen to form fibrin
• Fibrin creates covalent bonds and cross-linking of fibers create a meshwork in all directions of blood cells, platelets and plasma which adhere to the surface of damaged blood vessel
• After a clot is formed, the actin and myosin of the platelets trapped in the fibrin mesh interact in a manner like that in muscle contraction
• Ex: stroke tx window is BEFORE it’s stronger, before it contracts down

Question 37

cobwebs and clotting

Answer 37

• In traditional European medicine, cobwebs were used on wounds and cuts and seem to help healing and reduce bleeding. Spider webs are rich in vitamin K, which can be effective in clotting blood. Webs were used several hundred years ago as gauze pads to stop an injured person’s bleeding.

Question 38

dissolution of blood clot

Answer 38

• Clot lysis occurs when plasminogen (normally found in the blood) is activated to plasmin
• Activation occurs by tissue plasminogen activator (t-PA) released from the tissue, vascular endothelium, plasma, and urine (urokinase)
• Plasmin is an enzyme (resembles trypsin in pancreatic secretions) which digests fibrin fibers, fibrinogen, Factor V, Factor VIII, and Factor XII

Question 39

schematic pictures of fibrinolysis, describe it

Answer 39

plasminogen

↓

plasmin

(tPA ↑s this rxn, antifibrinolytics like TXA or amicar ↓s this rxn)

↓

fibrin(ogen) → fibrin(ogen) degradation products**

**this is why ppl with DIC have ↑ fibrin degrad. products! everything else is low, but FDP is high bc they’re breaking down clots over and over again!

Question 40

tissue plasminogen activator

Answer 40

exogenous plasminogen activators such as streptokinase are used to dissolve intravascular clots

-use in thrombotic stroke

Question 41

thrombolytics

what do they do?

when do they work best?

Answer 41

• Possess inherent fibrinolytic effects or enhances the body’s fibrinolytic system by converting endogenous pro-enzyme plasminogen to the fibrinolytic enzyme plasmin
• More capable of dissolving newly formed clots (platelet rich and weaker fibrinogen bonds)
• Older clots have more cross-linking and are more compacted = more difficult to dissolve

Question 42

POC coag measures:

PT

PTT

Answer 42

• Prothrombin time (PT) - evaluates the extrinsic pathway. A sample of blood plasma is incubated with tissue factor in the presence of excess Ca2+ . It is particularly sensitive to three of the four Vit-K dependent factors (II, VII and X). Commercial prothrombin reagents vary markedly in their responsiveness to warfarin-induced decreases in clotting factors and are not interchangeable between laboratories, therefore the INR is used for standardization
• Partial thromboplastin time (PTT) – indicates the performance of the intrinsic pathway. A sample of blood is triggered by adding an activator surface ( e.g., silica) plus phospholipid and CA2+

Question 43

POC coag measures:

ACT

TEG/ROTEM (viscoelastic testing)

Answer 43

• ACT - Performed by mixing whole blood with an activated substance to initiate activation of the clotting cascade. Widely used and is reliable for high heparin concentrations. Influenced by hypothermia, thrombocytopenia, coagulation deficiencies
• Viscoelastic Testing – Thromboelastometry (TEG) & Rotational Thromboelastometry (ROTEM) – a global assay for whole blood clotting including coagulation factors, inhibitors, anticoagulant drugs, platelets, and fibrinolysis

Question 44

POC coag measures:

bldg time

heparin [] measurements

plt fn tests

Answer 44

• Bleeding time – sensitive test of platelet function. A small standardized incision is made in the underside of the forearm and the amount of time it takes for bleeding to stop is recorded
  
  • old-timey
• Heparin Concentration Measurements – increasing concentrations of protamine are added to samples of heparin-containing blood. Time to clot is measured the sample in which heparin and protamine are most closely matched will clot first. (1mg Protamine will inhibit 1mg (100 units) heparin)
  
  • issue with standardization
• Platelet Function Tests - The classic method involves centrifugation of patient blood to obtain platelet- rich plasma, which is then analyzed in a cuvette at 37°C placed between a light source and photocell.
  
  • bc CBC tells you the number but not if they’re working

Question 45

Coagulation Test Normals:

bldg time

plt count

PT

Answer 45

bldg time: 3-10 min

plt count: 150-350,000 mm3

PT 12-14 sec

(prothrombin time)

Question 46

Coagulation Test Normals:

INR

aPTT (activ partial thromboplastin time)

TT (thrombin time)

Answer 46

INR: 0.9-1.2

apTT 25-35 sec

thrombin time 25-35 sec

Question 47

Coagulation Test Normals:

ACT (activ coagulation time)

Fibrinogen

Answer 47

ACT 80-150 sec

Fibrinogen >150 mg/dL

Remember all of them, but DEF the ACT. But all of them LOL.

Question 48

List some of the granular contents that plts release, which cause recruitment and activation of additional plts.

(dense granule and alpha granule)

total of 12 granules

Answer 48

• dense granules
  
  • ADP
  • ATP
  • serotonin
  • histamine
  • Ca++
• alpha granules
  
  • vWF
  • fibrinogen
  • F5, F7, F11, F13
  • PAI-1
  • TFPI
  • thrombospondin
  • fibronectin