1. From Blood to Host Defense Hemostasis and Clotting
Gregory J. Bagby, Ph.D.
Office: 310 (CSRB)

2. From Blood to Host Defense
Components and function
Hemostasis and clotting
The host defense system
General overview
Innate immune system
pathogen recognition
inflammatory response
Adaptive immune system
Humoral immune system and antibodies
Cell-mediated immune system

3. Definition Hemostasis is the prevention of blood loss by arresting of bleeding from injured vessels

4. Hemostasis and Its Control
Most effective with small vessels.
The greater intravascular pressure the harder it is to prevent blood loss.
Hematoma - accumulation of blood in tissue.
Three processes to decrease blood loss.
Vasoconstriction
Platelet aggregation
Blood coagulation or clotting
Steps to inhibit clot formation and dissolve clots once formed
Anticlotting system
Fibrinolytic system

5. Steps in Hemostasis following vessel injury: Vasoconstriction
Only effective in the smallest of vessels.
Injury leads to local contraction of vascular smooth muscle.
Mechanism unclear – endothelial cell disruption, mechanical stimulation, local mediators (EC, perivascular neurons)
Constriction presses opposed endothelial surfaces together
Contact induces stickiness between surfaces – adhesion molecules bind/adhere
Decreases blood flow to prolong contact time of damaged vessel with platelets

6. Steps in Hemostasis following Vessel Injury: Platelet Aggregation
Plasma von Willebrand factor binds to collagen which binds to platelets
Platelets degranulate to release mediators
ADP and thromboxane A2 - amplifies platelet adherence and degranulation.
Serotonin – stimulates constriction
Thromboplastin – stimulates coagulation
Plasma fibrinogen – bridges between platelets (aggregation)
Thromboxane A2 (TXA2) is a thromboxane. It is produced by activated platelets and has prothrombotic properties: it stimulates activation of new platelets as well as increases platelet aggregation. This is achieved by mediating expression of the glycoprotein complex GP IIb/IIIa in the cell membrane of platelets. Circulating fibrinogen binds these receptors on adjacent platelets, further strengthening the clot.

7. Inhibition of Platelet Aggregation
Adjacent endothelial cells produce agents that inhibit platelet activation/aggregation and cause vasodilatation:
Prostacycyclin (PGI2) – Inhibits platelet function
Nitric oxide - Vasodilator

8. Steps in Hemostasis following vessel injury: Coagulation
Blood coagulation – transformation of blood into a gel (clot or thrombus)
Main ingredient – a polymer of fibrin that forms a mesh
Local response around a platelet plug
Complex process involving sequential re-enforced activation of plasma factors (proteins)
Key step in clotting pathway is production of the enzyme thrombin
Thrombin cleaves fibrinogen to fibrin (soluble) which bind together to form loose mesh
Thrombin activates Factor XIII which covalently cross-links fibrins to stabilize the fibrin network
Thrombin activates clotting pathway leading to own production

9. The clotting pathway or cascade involves about 15 proteins.
Thrombin – FII a serine protease
FXIII (fibrin stabilizing factor) crosslinks fibrin which also binds to platelets
The clotting pathway or cascade involves about 15 proteins.
Thrombin plays directly initiates clot formation by catalyzing the conversion of fibrinogen to fibrin, and serving as a positive feedback modulator of the cascade.

10. Platelets & Calcium Important in Clot Formation
Blood cells trapped in the fibrin meshwork, but not essential
Clot possible in absence of cellular elements except platelets.
Thrombin activates platelets in addition to vessel injury
Activated platelets express receptors that bind several of the clotting factors
Allows reactions to occur on the surface platelets
Platelet factor (phospholipid) serves as a cofactor for clotting pathway reactions
Calcium also required for several steps (always sufficient)

11. The Clotting Cascade from Injury to Thrombin Formation
Two pathways leading to pro-thrombin to thrombin reaction
Intrinsic pathway – constituents all in the blood
Extrinsic pathway – cellular elements outside blood are needed
Two pathways activated in sequence with thrombin serving as the link between them.

12. Clotting Cascade Intrinsic pathway Extrinsic pathway
Collagen activated
Inactive to active proteases
Cofactors (VIIIa & Va)
Extrinsic pathway
Cell-assoc Tissue Factor
Thrombin produced by this pathway activates factors in the intrinsic pathway.
Results in more thrombin to accelerate fibrin formation
Tissue factor = FIII
Activated platelets release thomboplastic factor (PF3)
Active factor X, along with factor III, factor V, Ca++, and platelet thromboplastic factor (PF3), will activate prothrombin activator which converts prothrombin to thrombin.

13. Procoagulant Actions of Thrombin
Cleaves fibrinogen to fibrin
Activates clotting factors
Factor XIII to cross-link fibrin
Intrinsic pathway via factor XI
Important co-factors VIII and V
Stimulates platelet activation

14. Hemophilia Mostly genetic X-linked disorders seen primarily in men.
Hemophilias
Hemophilia A or classical hemophilia - Lack of factor VIII (~77% of cases).
Hemophilia B - Lack of factor IX.
Hemophilia C (both sexes) – Lack of factor XI. Mild because IX (which is activated by XI) can also be activated by VII.
Symptoms in life and disabling.
Bleeds impair joint function.
Treated by transfusions of plasma and injections of purified clotting factors (expensive to treat and inconvenient to the patient).
Surgery, simple dental procedures - life-threatening.

15. Liver Plays Important Indirect Role in Clotting
Site of clotting factor synthesis
Vitamin K
Bile salts produced by liver important vitamin K, a lipid soluble vitamin, absorption
Vitamin K important for clotting factor synthesis
Prothrombin
Other clotting factors
Liver failure – results in sever bleeding problems

16. An Endogenous Anticlotting and Clot Lysing System Prevent, Limit or Remove Clots
Endogenous anticlotting system - Factors that oppose and limit clot formation.
The fibrinolytic system – dissolves clots after they are formed.
Anticlotting drugs – Used to treat patients to decrease clotting or dissolve clots that inappropriately stop blood flow to important tissue (heart, brain).

17. Three Factors that Oppose Clot Formation
Plasma tissue factor pathway inhibitor (TFPI) binds to and inhibits tissue factor-factor VIIa complexes to inhibit continuous production of factor Xa
Extrinsic pathway only generates small amount of thrombin
Thrombin binding to thrombomodulin on endothelial cells
Inactivates thrombins clot-producing effects
Bound thrombin binds to plasma protein C which inactivates Factors VIIIa and V
Thrombin indirectly inactivates clotting
Plasma antithrombin III
Inactivates thrombin and other clotting factors
Works via binding to heparin on endothelial cells
Prevents spread of the clot to uninjured areas

18. Fibrinolytic System Removes Clots as Part of the Repair Process
It’s as complex as clotting with activators and inhibitors
Plasminogen activators, important e.g. is t-Pa
Plasminogen activators like Tissue Plasminogen Activator (t-PA)
Plasminogen
Plasmin
Fibrin
Soluble Fibrin Fragments
Synthetic plasminogen activators can be used
immediately after a stroke or heart attack
to help dissolve clots and restore blood flow.

19. Anticlotting Roles of Endothelial Cells
Barrier between blood and subendothelial extracellular matrix and cells – inhibits platelet aggregation & formation of TF-VIIa complexes
Synthesis of PGI2 and nitric oxide – inhibits platelet activation and aggregation
Secretes tissue factor pathway inhibitor – inhibits TF-factor VIIa complexes to generate Xa
Binds thrombin via thrombomodulin to activate protein C – inactivates factors VIIIa and Va
Display heparin on surface – binds antithrombin III to inactive thrombin and other clotting factors
Secretes tissue plasminogen factor – catalyzes formation of plasmin to dissolve clots

20. Anticlotting and Thrombolytic Drugs
Agents that depress the action of procoagulants
Aspirin – platelet cyclooxygenase inhibitor.
Oral anticoagulants (Dicumarol) – inhibits action of vitamin K required for synthesis of clotting factors by the liver.
Heparin – activates antithrombin III.
Fibrinogen blockers - interfere with fibrinogen binding to platelets.
Agents that dissolve clots (thrombolytic drugs)
Recombinant t-PA
Desmodus rotundus salivary plasmninogen activator (DSPA)
Streptokinase
Aspirin – a COX-1 inhibiter – decreasing production of thromboxane A-2 by platelets
Aspirin acetylates COXes active site