Inflammation and Coagulation MDRs, Thrombosis MDR, and Cornell Coagulation Videos

Tissue Factor

Constitutively expressed in most cells within the body

• Highest concentrations in the brain and endothelial cells

What happens when tissue factor is exposed to the blood stream?

Exposed to FVII which circulates in its active form in the bloodstream

• This complex converts FX to FXa which mediates the conversion of FII to FIIa (thrombin)

What stimulates the expression of TF on the surface of endothelial cells and mononuclear cells?

Inflammatory cytokines (particularly IL-6)

Actions of Thrombin in the Inflammation and Coagulation Systems

Facilitates the production of fibrin

Activates FV and FVIII, Protein C, and thrombin activatable fibrinolysis inhibitor (TAFI)

Most potent stimulator of platelets

Can stimulate stimulate leukocytes to produce inflammatory cytokines such as TNF-a, IL-1 and IL-6 through the NF-kB pathway

Can directly stimulate endothelial cells causing them to produce IL-6 and platelet activating factor (stimulates platelets and is a powerful chemoattractant)

Actions of TFPI

Endothelial protein

Main purpose is to decrease coagulation by binding the TF-FVII complex and inactivating it

Inhibits TF and IL-6 to prevent excessive inflammation and coagulation distant from site of injury

Actions of TAFI

Keep fibrin from being broken down (counteracts plasmin)

Inactivates complement 5a which ends up being anti-inflammatory

Action of Protease Activated Receptors (PARs) 1, 3, and 4

Bind thrombin

Action of Protease Activated Receptor (PAR) 2

Can be activated by the TF-VII complex and Fx

What happens once a substrate binds to a protease activated receptor?

The PAR cleaves part of the receptor exposing a neoamino terminus which allows the receptor to become its own ligand

Changes that Occur Once the Platelet is Activated that Propagate Inflammation and Coagulation

Upon activation, P-selectin is exposed on the platelet membrane (never expressed on a non-activated platelet)

• Allows the platelet to bind to endothelial cells and upon binding cause the endothelial cell surface to interact with leukocytes

• P-selectin can also bind to P-selectin glycoprotein-1 (PSGL-1) on the surface of leukocytes which once bound, causes activation of the target leukocyte, promoting TF expression from the leukocyte as well as reactive oxygen species (ROS) and cytokine release (IL-1, CD 40 ligand)

TLR-4 Receptor on Platelets

Platelets have a TLR-4 receptor which can be activated by LPS

LPS activated platelets tend to migrate to the point of infection and will engage with leukocytes to form neutrophil extracellular traps

LPS activated platelets degranulate and express P-selectin on its cell surface

When this platelet binds to the P-selectin glycoligand (PSGL) on a circulating neutrophil it induces NETosis

• Activated platelets can also secrete a damage associated molecular protein - high mobility group box-1 (HMGB-1) which can bind to neutrophil receptors and induce NETosis

NETosis

Leukocytes release neutrophil extracellular traps (NETs) composed of cell free DNA, histones, and antimicrobial proteases to form a web like structure capable of sequestering bacteria and destroying them

NETs can also cause damage to endothelium and surrounding cells causing vascular leakage and edema in the affected tissues

Endothelial Cells in Inflammation and Coagulation

In homeostasis, intact endothelial cells are essential to prevent soluble FVIIa in the blood from interacting with the TF underlying the cells

In patients with severe inflammation, various cytokines such as TNF and IL-1 can induce TF expression on endothelial cells

When tissue disruption occurs, endothelial cells are compromised allowing the FVII in the blood to adhere to TF within the cell or expressed ont eh endothelial cell surface after cell damage

Endothelial Protein C Receptor (EPCR)

Constitutively expressed protein on the endothelial cell surface that is a key regulator in balancing inflammation and coagulation

Binding of this receptor to circulating Protein C vastly increases its conversion to activated protein C (APC) and allows it to exert its anticoagulation and anti-inflammatory effects

EPCR can become soluble and when it does so it can complex with neutrophil proteinase-3 and integrins to block adhesios of leukocytes with the endothelial cells

Thrombomodulin

In homeostasis, this protein is constitutively expressed to bind excessive thrombin away from the site of injury

When thrombin is bound to endothelial thrombomodulin not only does it prevent thrombin from converting fibrinogen to fibrin, but it also allows the conversion of circulating protein C to APC

What activates Protein C complex?

Thrombin-thrombomodulin complex

Actions of Activated Protein C (APC)

Perhaps the most important player in decreasing both excessive inflammation and hypercoagulation

Anti-coagulant properties

• After binding to cofactor Protein S it can inactivate FV and FVIII

Interacts directly with EPCR which enhances the conversion of protein C to APC by 5-fold

• Interaction between APC and EPCR also blocks intracellular NF-kB which prevents cells from forming and releasing cytokines and chemokines

Inhibits adhesion of leukocytes to endothelial cells

Can inhibit endothelial cell apoptosis likely through binding to EPCR and possibly PAR-1 on the endothelial cells

Soluble EPCR-APC Complex

Soluble EPCR-APC complex can act at sites distant to the site of injury

Sepsis decreases the ability of EPCR-APC to become soluble and exert its anti-inflammatory and anti-coagulation effects systemically

Actions of Antithrombin

Antithrombotic protein

Binds to thrombin and prevents its pro-coagulant activation

Can also cause prostacyclin release from endothelial cells

• Prostacyclin inhibits platelet activation and aggregation and has effects on endothelial cells such as reduction in tethering of neutrophils and decreased secretion of cytokines

• When antithrombin binds to leukocytes through receptors like syndecan-4, it can block the interaction of these cells with endothelial cells

  • Accomplishes this by decreasing the expression of P-selectin, reducing activation of leukocytes and/or increase prostacyclin release

Procoagulant Components of Hemostasis

An activated vascular endothelium, activated platelets, and coagulation proteins

Abnormal or decreased fibrinolysis

Anticoagulant Components of Hemostasis

Antithrombin (AT), protein C and protein S (PC/PS), factor V, and prostacyclin (PGI2)

Normal fibrinolysis is antithrombotic

What are the two types of physiologic plasminogen activators?

Tissue plasminogen activator (tPA)

Urokinase plasminogen activator (uPA)

Tissue Plasminogen Activator

Released from endothelial cells in response to direct injury or following stimulation by thrombin

Urokinase Plasminogen Activator

Found in numerous cell types in the body and may have a greater role in extravascular fibrinolysis

What is the main regulator of the initiation phase of coagulation?

Tissue factor pathway inhibitor (TFPI)

What are the main regulators of the propagation phase of coagulation?

Antithrombin (AT) and activated protein C (aPC) pathways

Where are the three pools of TFPI in the body?

Within microvascular endothelial cells, stored in platelets, and circulating bound to lipoproteins

Actions of TFPI

Inactivates both the TF-FVIIa complex and FXa

Initially TFPI complexes with and inactivates FXa

• The TFPI-FXa complex then binds to the TF-FVIIa complex forming a tetrameter than inactivates the TF-VIIa

What is the half-life of antithrombin in plasma?

Approximately 48-56 hours

May decrease to as little as 8-12 hours in sepsis induced consumptive coagulopathy

Actions of Antithrombin

Specific thrombin (FII) and FXa inhibitor, but also inhibits FVIIa, XIa, XIIa, and kallikrein

AT binds to serine protease coagulation factors via a serine-binding domain and the complex is removed by the reticuloendothelial system

When does AT function most effectively?

When its heparin binding domain is also occupied

• Binding of cell surface heparin and other glycosaminoglycans (GAGs) results in a conformational change that allosterically potentiates AT enzymatic activity great than 1000 fold

Anti-Inflammatory Functions of AT Bound to GAGs

By damping down coagulation, it reduces the pro-inflammatory effects of the downstream coagulation factors such as thrombin

Also block expression of NF-kappa B and thereby reduces cytokine production and decreases leukocyte activation and adhesion

Also increases PGI2 production

Anti-inflammatory properites not realized when AT is bound to exogenously administered heparins

Where is Protein C synthesized?

In an inactive form in the liver, in a vitamin K dependent process

How is protein C activated?

Protein C is converted to its active form, aPC in the presence of the thrombin + thrombomodulin complex, a process which is facilitated by the endothelial protein C receptor

Action of Protein C

Once activated, protein C dissociated from the endothelial protein C receptor, binds to protein S and this complex inactivates FVa and FVIIIa

• Anti-coagulant action

Inhibits plasminogen activator inhibitor-1 (PAI-1)

• Profibrinolytic

Inhibits pro-inflammatory cytokine production and leukocyte activation likely via downregulation of NF-kB

• Anti-inflammatory

Role of Thrombomodulin

Competes with the thrombin receptor for thrombin binding, minimizing the procoagulant and proinflammatory effects of thrombin

What activates thrombin-activatable fibrinolysis inhibitor (TAFI)?

Thrombin, thrombin-TM complexes, and plasmin

What is the action of TAFI?

Attenuates the conversion of plasminogen to plasmin

When thrombin is bound to thrombomodulin, activation of TAFI, and hence inhibition of fibrinolysis is increased 1250 times

What comprises Virchow’s triad?

Blood stasis

Hypercoagulability

Endothelial damage

What characterizes hypercoagulability?

Increased platelet activation, enhanced activation of coagulation factors, reduction of natural anticoagulant or inhibition of fibrinolysis

What can cause blood stasis?

Reduced blood velocity, reduced or turbulent blood flow, often caused by vascular or heart valve or chamber anomalies

What does endothelial injury revolve around?

Increased platelet activation and increased concentration of activated clotting factors

Composition of Venous Thrombus

Primarily composed of red blood cells embedded within a fibrin meshwork as they are formed at low shear stress

What is the most important part of arterial thrombus formation?

Platelet adhesion to the endothelium due to the predominance of high shear stress conditions

How does aspirin work?

Acts on the arachidonic acid pathway and irreversibly inhibits the COX pathways

• COX1 and COX2 catalyze the conversion or arachidonic acid to PGH2, the precursor for several metabolites including thromboxane A2 (TXA2)

What is the function of thromboxane A2?

Enhances platelet function and promotes platelet aggregation and vasoconstriction

MOA of Clopidogrel

Thienopyridine that selectively inhibits ADP-induced platelet aggregation

Has no direct effects on arachidonic acid metabolism

Requires hepatic biotransformation to produce the active metabolite

MOA of Heparin

Complexes with and catalyzes the activity of the anticoagulant protein antithrombin

Action of the Heparin:Antithrombin Complex

Inhibits coagulation factors IIa (thrombin), IXa, Xa, Xia, and XIIa

• IIa and Xa are most sensitive

By inactivating thrombin, heparin not only prevents fibrin formation, but inhibits thrombin-induced platelet activation and continued activation of coagulation factors V and VIII

What fraction of unfractionated heparin molecules contain the binding site for antithrombin?

1/3

The other 2/3 have minimal anticoagulant activity

Monitoring for Unfractionated Heparin Therapy

aPTT with an accepted therapeutic target range of 1.5-2.5 times the normal control aPTT value

How is the action of LMWH different from UFH?

LMWH has reduced anti-IIa activity relative to anti-Xa activity

MOA of LMWH

Binds to and catalyzes the activity of antithrombin

Can you monitor LMWH with aPTT?

No, prolonged aPTT seen with UFH primarily reflects inhibition of factor IIa

• Inhibition of Xa has little effect on aPTT

What is the recommended monitoring for LMWH therapy?

Chromogenic assays of anti-factor Xa activity

Clinical Signs Associated with PTE

Variable, from mild dyspnea to sudden death, but revolve around respiratory signs such as hypoxemia, dyspnea, and hyperventilation

• Hypoxemia is secondary to low V/Q matching

Arterial Blood Gas Findings with PTE

100% of dogs with PTE had an increased alveolar-arterial gradient and almost half had hyperventilation

Radiographic Patterns Described with PTE

Westermark sign - regional oligemia (hypoperfusion)

Hampton hump sign - shallow wedge-shaped opacity in the periphery of the lung with its base against the pleural surface

Routine Coagulation Profiles in PTE

Typically normal

D-dimers are more indicative that thrombosis has occurred

Treatment for PTE

Symptomatic treatment (oxygen, mechanical ventilation), treatment of the cause, and trhombophylaxis (UF, LMWH, or anti-XA) is usually provided

Effects of Thrombin on Platelets

Interact with PAR1 endothelium

• In the presence of thrombin, platelets will bind more tightly to the endothelium

Activate platelets (degranulate alpha granules, activate GpIIb)

• GpIIb likes to bind with the next platelet (II says that it needs the next platelet in line)

Scramblase moves phosphatidylserine (PS) from inner to outer platelet membrane → negatively charged phospholipid surface

What are FDPs?

Breakdown products of fibrin, crosslinked fibrin, and fibrinogen

Doesn’t give evidence that a clot was present

What are D-dimers?

Breakdown products of crosslinked fibrin

Tells you a clot was present

What are the actions of thrombin? Are they pro-clot or anti-clot?

Pro-clot

• Activates platelets to propagate coagulation

• Feeding back and activating FXI to amplify coagulation

• Making fibrin and crosslinking it

• Activates TAFI - inhibits fibrinolysis

Anti-clot

• Activates tPA → plasmin

• Binds to thrombomodulin → activates protein C

Thrombin activatable fibrinolysis inhibitor (TAFI)

Removes lysine residues from fibrin clot

Plasmin will not bind to the clot

Activated by thrombin

a-2 antiplasmin

Inhibits plasmin

Plasminogen Activator-Inhibitor (PAI-1)

Inhibits activity of tPA/uPA → no plasmin formation

Tissue Factor Pathway Inhibitor (TFPI)

Inhibits Xa, TF-VIIa complex (extrinsic tenase complex)

Released from endothelium in response to heparin

Stored in platelets, endothelial cells

Circulating (bound to lipoproteins)

Protein C/Protein S

II binds to thrombomodulin on endothelial cell

• Activates protein C which is bound to the endothelial protein C receptor

• Becomes APC

• Goes down the endothelium and activates protein S and have activated C and S

Inactivates V and VIII

• Can remember because S kind of looks like 5 and can easily make an S into an 8

a-1 Protease Inhibitor

Inactivates Xa

a-2 Macroglobulin

Inactivates II

Heparan Sulfate

Proteoglycan on glycocalyx

Works with anti-thrombin

Inhibits II and X

Antithrombin

Made in the liver

Circulates in plasma

Inhibits II and X

Unfractionated Heparin

Potentiates antithrombin

• AT → inhibits II and X

• UFH binds to antithrombin and changes its shape so it can quickly and easily bind FX

• Thrombin is a little bigger and doesn't find so well in the notch of antithrombin, with thrombin you need the tail of UFH to come and lock in thrombin

• If you're binding FX and the long arm is still free the long arm that isn't locking in it can bind IX, XI, VII, and XII

• Some inactivation of IX, XI, VII, XII

• Will cause release of TFPI from the endothelium

What % of UFH is the pentasaccharide that can activate AT?

20-50%

How is unfractionated heparin cleared?

Fast macrophages, endothelial cell binding → depolymerization

Slow clearance kidneys

Non linear clearance

What is the duration of action of unfractionated heparin?

Short duration of action (60 minutes)

Unfractionated Heparin Reversal

Protamine sulfate

Low Molecular Weight Heparin

Only binds to X

No long tail → no binding to II

What fraction of the size of UFH is LMWH?

1/3 size (4-5000 daltons)

What % of the size of UFH is LMWH?

50-75%

Characteristics of LMWH

100% SQ bioavailability

More predictable effects

Longer half life (less binding macrophages, endothelium)

Clearance kidneys

Protamine zinc reverses 60% of anti-Xa activity (favors larger chains)

Ultra Low Molecular Weight Heparin

Fondaparinux

<3000 daltons

Only inhibit Xa + AT

• No other effects of heparin

• Only renal clearance

Therapeutic Monitoring for UFH

Monitor with aPTT

1.5-2.5x mean aPTT

Large multimers degrade faster

aPTT does not tell you about FX activity

• Because it takes longer for small pieces to break down aPTT can be normal and still have FX inhibition

Univalent Direct Thrombin Inhibitors

e.g. argatroban, dabigatran

Non-covalent binding to thrombin

Reversible

Minimal bleeding risk and fast return to baseline

Can be reversed

Bivalent Direct Thrombin Inhibitors

e.g. lepirudine, desirudine, hirudin

Irreversible binding to thrombin

Cleaved by II once bound → slow return to function

Factor Xa Inhibitors

Primary site of action is the amplification stage of coagulation

No effects on platelets

Directly binds to Xa (bound and free)

Reversible

What is the major goal of platelet activation?

Activation of the integrin IIb/IIIa receptor → binds fibrinogen → platelets aggregate

Platelet GPCR Thrombin Receptors

PAR1

PAR4

PAR1 Ligand

Thrombin

PAR4 Ligand

Thrombin

Platelet ADP Receptors

P2Y₁

P2Y₁₂

P2Y1 Ligand

ADP

P2Y12 Ligand

ADP

Platelet Prostaglandin Family Receptors

TXA2

TXA2 Ligand

TXA2

Antiplatelet Drugs that Interfere with GPCRs and Their MOAs

COX Inhibitors (aspirin) - decrease thromboxane A2 production

Thienopyridines (clopidogrel, ticlopidine, prasugrel) - pro-drugs, irreversibly bind at P2Y12 receptors

Nucleoside analogs (cangrelor, ticagrelor) - reversibly block P2Y12 receptors

Platelet Leucine Rich Receptors

GPIb-IX-V

TLR2

TLR4

TLR9

Action of GPIb-IX-V Receptor

• GPIb-IX-V is the major platelet receptor for vWF

  • Controls the first step in platelet adhesion, attaches the vWF on damaged subendothelium

  • In a high shear stress state, increased affinity for vWF, attachment cycles on and off rapidly so platelet can roll over the endothelial surface by temporary tethering

  • Have short cytoplasmic tails so participate in signal transduction and regulate binding affinity

  • Binding trigger intracellular signaling events, platelet activation, adhesion, aggregation

Platelet GP1b-IX-V Receptor Ligands

vWF, collagen, P-selectin