Inflammatio

Question 1

Steroids and Demargination

(Neutrophils)

Answer 1

Neutrophils bind to the endothelial cells by rolling adhesion made possible by L-Selectins. These neutrophils are marginalized. Steriods stop the transcription of L-Selectins causing the neutrophil to fall off the endothelial cells into the circulation, called demargination.

Neutrophils adhere to the endothelium by L(Leukocyte)-selectin and are not free flowing in the blood. The pool of neutrophils adhered to the endothelium are the marginated neutrophils.

Note: Selectins are CAMs (Cell Adhesion Molecules). Selectins are lectins, a cell adhesion protein that binds to carbohydrates. Three classes of selectins are L(Leukocyte), P(Platelet, Endothelial,Leukoytes), and E(Endothelial).

L-Selectin is a cell surface receptor that binds to endothelial receptors for adherance and rolling. The L-Selectin are in constant turn over as the neutrophil rolls along the endothelium. Sheddase removes the L-Selectin from the suface of the neutrophil. A new L-Selectin replenishes the removed one.

Steroids block the transcription of L-selectin. Since there are no L-Selectins to replace the ones removed by Sheddase, rolling cannor occur and the Neutrophils become free flowing in the blood in a process called demarginalization.

Question 2

Platelet Adhesion/Activation/Aggregation and Drugs

Answer 2

Adhesion – Activation – Aggregation

Collagen is exposed to the platelets in damaged blood vessel walls. Integrins and glycoprotein receptors bind to the collagen, but not sufficient for high sheer stress. A platelet glycoprotein complex binds vWF then to collagen in order to bind sufficient to overcome high sheer stress. vWF is a multmeric protein found in blood plasma bound to factor VIII and released from factor VIII by thrombin – see activation of thrombin below through TF. vWF is produced constitutively in endothelium and stored in endothelium Wiebel Palade bodies, and in alpha granules of platelets. ADP and TXA2 are released from platelet granules. Thrombin (IIa), ADP and TXA2 bind to their associated receptors – TP, P2Y and PAR – causing the activation of a high affinity integrin. Once activated, the high affinity integrin binds to fibinogen and vWF to form crosslinks between platelets.

Coagulation

Note: P2Y (Purinogenic G-coupled protein receptor), TP (Thromboxane receptor, G-coupled protein receptor), PAR (Protease Activated Receptor).

Note: ADAMTS13 protease inactivates vWF. Deficiencies of ADAMTS13 had been found in Thrombotic Thrombocytopenia Purpura (TTP), and Hemolytic Uremic Syndrome (HUS). In TTP and HUS the platelets get used up as vWF is not inactivated. Also, the microcloting of platelets sheers red blood cells causing hemolysis.

Note: Tissue Factor (TF) is expressed on the surface of fibroblasts, smooth muscle cells (SMCs) and on activated endothelium and activated monocytes during inflammation processes (when activated by TNFalpha). TF is not normally exposed to blood. Factor VII (circulating in the blood) binds to TF on the activated endothelium, fibroblasts and SMCs, and is activated to form TF/VIIa. VIIa then activates factor IX into IXa, and factor X into Xa, leading to the activation of factor II into IIa (thrombin). Thrombin converts fibrinogen into fibrin, forming the fibrin mesh in coagulation.

**TF/VIIa activates the proteases Factor IX into IXa, and X into Xa leading to the formation of Thrombin which forms the Fibrin mesh.

Note: Thromboplastin is the combination of TF and phospholipids. Thromboplastin is needed to activate the extrinsic pathway. Phospholipids were later termed the partial thromboplastin and activate the intrinsic pathway, TF is not needed to activate the intrinsic pathway.

Adhesion
Collagen exposed. Integrin GPIa-IIa and then GP receptor GPVI bind to collagen for low sheer adhesion. GPIb-V-IX complex binds to vWF bound to collagen leading to tight adhesion under high sheer stress.

When collagen is exposed to the blood, platelets bind to collagen by the integrin GPIa-IIa first, which then allows GPVI receptor binding(glycoprotein receptor for collagen). The binding stabilizes the platelets but not sufficient under high sheer stress.

Note: Integrins are proteins that function mechanically, by attaching the cell cytoskeleton to the extracellular matrix (ECM), and biochemically, by sensing whether adhesion has occurred through intracellular cascades.

To ensure platelet adhesion under high sheer stress, vWF binds to the glycoprotein complex GPIb-V-IX, and to collagen.

Activation
ADP and TXA2 released through granules.
Once platelets are adhered by integrins and glycoproteins, intracellular cascades cause a change in the cytoskeleton in addition to the release contents stored in granuales – alpha granuales and dense granules. **ADP and TXA2 are stored in granuales and releaed.

• *Aggregation**
• *Integrin GPIIb-IIIa is activated to a high affinity integrin through the binding of IIa, ADP, TXA2 binding to their corresponding receptors. Once activated, fibrinogen and vWF can bind to GPIIb-IIIa creating crossbridges to other platelets.**

Note: **ADP and TXA2 are platelet aggregating agents by binding to platelet membrane receptors. Medications used against these receptors (ASA, Plavix) leads to less platelet adhesion.

**Thrombin (IIa) is the **most potent platelet aggregating agent and strongest agonist **. When II binds to platelet membrane receptors, PARs, it is is converted to IIa on the activated platelet surface.

By binding of ADP, TXA2, IIb, to their receptors P2Y, TP, PAR, causes integrin GPIIb-IIIa to be converted to a high affinity state. Fibrinogen and vWF binds to the high affinitin integrin GPIIb-IIIa creating crossbridges to other platelets, by vWF and fibrinogen binding to GPIb-V-IX and GPIIb-IIIa on the other platelets.

• *Note: vWF** is produced constitutively by endothelium and circulates in the blood bound to Factor VIII in a vWF/VIII complex. Factor VIII is degraded when not bound by vWF. vWF is also produced and stored in endothelial cell granules called Weibel Palade bodies (along with P-Selectin), and found in platelets in alpha granules.
• ***vWF is needed for platelet high sheer stress adhesion and platelet aggregation.**
• *Medications**
• *DDAVP (Desmopressin-synthetic Vasopressin).** Enhances release of vWF from endothelial cell granules.

ASA. Blocks COX1 and formation of TXA2. TXA2 cannot bind to TP. Limits the activation of high affinity integrin GPIIb-IIIa and subsequent binding of fibrinogen and vWF to create crossbridges.

Unfractionated Heparin. Sequesters thrombin and factor Xa through the activation of anti-thrombin (AT) and limits the binding of thrombin to PAR. Limits the activation of high affinity integrin GPIIb-IIIa and subsequent binding of fibrinogen and vWF to create crossbridges.

Clopidogrel {Plavix}. ADP receptor P2Y (Purinogenic G-coupled protein receptor) antongonist. Limits activation of the high affinity integrin GPIIb-IIIa and subsequent binding of fibrinogen and vWF to create crossbridges.

Adciximab (ReoPro) and Eptifibatide (Integrilin). Blocks GPII-IIIa receptors.

Question 3

Coagulation Cascade and Drugs

Answer 3

Unfractionated Heparin (works same as endogenous heparin) - Inhibits Thrombin (IIa) and Xa on a 1:1 ratio. Heparin binds to Anti-thrombin-thrombin complex to potentiate anti-thrombin effect. When anti-thrombin levels are low (in procoagulant states like septic shock) heparin resistance may develop. Thrombin inhibition prolongs PTT. PTT is used to monitor the anticoagulation effect because variable levels of anticoagulation may be seen despite consistent weight based dosing. Protamine is used to reverse the effects of heparin, where it binds to heparin or LMWH to form an ion pair that does not allow for anti-coagulant activity.

LMWH (Enoxaparin, {Lovenox}) – inhibits Xa more than IIa due to the size of the molecule, in a ratio of 2:1 to 4:1 with minimal effect on aPTT. Monitoring is not required because weight based therapy produces consistent levels of anti-coagulation.

Fondaparinux – is a LMWH that activates antithrombin so only Xa is inhibited.

HIT – Heparin Induced Thrombocytopenia – Heparin binds to Platelet Factor 4 (PF4) and becomes immunogenic (heparin acts as a hapten) with IgG, IgM or IgA antibodies. The Fc portion of the Heparin/PF4/IgG binds to a Fc repector on platelets. This leads to the activation of platelets, the formation of platelet microparticles and blood clots, resulting in the thrombocytopenia. The drop in platelets occurs ~10days after exposure to heparin. Clinical findings include: drop in platelets >50 percent 5-10days after initiation of unfractionated heparin or LMWH, thrombosis if present is the most serious finding.

Danaparoid – considered a LMWH however is chemically distinct from heparin, has different protein binding properties, and thus has little cross reactivity to heparin. Used in heparin intolerant patients with HIT.

Apixaban {Eliquis}, Rivaroxaban {Xeralto} – Xa inhibitors.

Dabigatran {Pradaxa} – IIa (Thrombin) inhibitor.

Question 4

Antiphospholipid Syndrome

Answer 4

Clinical suspician when:

• *1. Unexplained venous or arterial thrombus especially in young adults.**
• *2. Adverse events related to pregnancy**, including fetal death after 10 weeks gestation, premature birth due to severe preeclampsia or placental insufficiency, or multiple embryonic losses (<10 weeks gestation).
• *3. Unexplained thrombcytopenia or prolonged aPTT.**

A systemic autoimmune disease also raises suspicion especially with SLE (Systemic Lupus Erythematosus).

INVESTIGATIO]
L(H)/Immun] Anticardiolipin antibody (ELISA)
Anti-beta2-glycoprotein (GP) 1 (ELISA)
Lupus Anticoagulant (LA).

Question 5

Diapedesis

Leukocyte Extravasation

Answer 5

Chemoattraction – rolling adhesion – tight adhesion – transmigration

Leukocyte extravasation occurs at the post-capillary venules where hemodynamic sheer forces are minimised. Macrophases release cytokines activating endothelial cells. P-Selectins are brought to the surface of the endothelial cells. Leukocyte glycoproteins bind to P-Selectins. Leukocyte Integrins are activated and bind to endothelial glycoproteins causing them to be converted from a low affinity state to a high affinity state. Leukocyte changes the cytoskeleton to allow for movement through the endothelial cells.

• *Chemoattraction**
• *TNFalpha, IL-1.**

Macrophages release chemokines TNFalpha, IL-1. TNFalpha, IL-1 cause endothelial cells to bring P-Selectins on their surface from stored granules, and leukocytes are localized to the released chemokines.

Note: P-Selectins and vWF are stored in endothelial storage granules called Weibel-Palade bodies relased upon exposure to TNFalpha.

• *Rolling Adhesion**
• *Glycoprotein on leukocyte binds to P-Selectin on endothelial cells.**

Like velco, PSGL-1 on the leukocytes bind to P-Selectins on the surface of the endothelium with marginal affinity. PSGL-1 (P-Selectin Glycoprotein Ligand-1).

Note: Selectins are CAMs (Cell Adhesion Molecules) made of lectins, a cell adhesion protein that binds to carbohydrates. Three classes of Selectins are L(Leukocyte)-Selectin, P(Platelet, Endothelial,Leukoytes)-Selectin, and E(Endothelial)-Selectin.

• *Tight Adhesion**
• *Integrins on leukocyte binds to glycoprotein on endothelial cell.**

Integrin ligands (ITGAM, LFA1) on leukocyte binds to ICAM (glycoprotein) and VCAM (glycoprotein) on endothelial cells. Leukocytes are activated through juxtacrine signalling, binding of integrin to ICAM/VCAM, and chemokines, changing Integrin from a low affinity state to a high affinity state. This immobilizes the leukocyte despite sheer forces in the blood.

Note: ITGAM, also called Mac-1, (Integrin Alpha M), LFA1 (Lymphocyte function-associated antigen 1), ICAM (InterCellular Adhesion Molecule – glycoprotein) and VCAM (Vascular Cell Adhesion Molecule)

Note: Integrins are transmembrane receptors that create cell-cell and cell-ECM bridges causing intracellular signalling. Ligands for integrins are collagen, fibronectin, vitronectin, and laminin.

• *Transmigration**
• *Cytoskeleton changes** in the leukocyte and pseudopodia allow the leucocyte to be pulled through the endothelial gaps with the help of PECAM proteins.