1. CLOT FORMATION AND LYSIS
Function
Consequences of dysfunction
Rapid formation of mechanically sound clot at areas of injury
Bleeding
Prevent clotting in areas removed from injury
Thrombosis
Controlled clot lysis and replacement by connective tissue
Bleeding (if lysis too fast)
Thrombosis (if too slow)
Poor healing

3. COMPONENTS OF THE HEMOSTATIC SYSTEM
Function
Platelets and von Willebrand factor
Formation of primary hemostatic plug
Coagulation cascade
Formation of fibrin clot (stabilizes and strengthens hemostatic plug)
Vessel wall/endothelium
Endothelium antithrombotic; vasoconstriction limits bleeding from injured site
Fibrinolytic system
Controlled clot lysis allows wound healing
Defects in primary hemostasis tend to cause immediate bleeding after injury
Defects in fibrin formation tend to cause delayed bleeding
Endothelial damage promotes thrombosis
Uncontrolled fibrinolysis causes bleeding

4. PLATELETS

5. Platelets are required to seal areas of vessel injury
Albumin extravasation vs time in injured vessels after inhibition of platelets (left) and thrombin (right)
Blood 2015;127:1598

6. PLATELETS 150-400,000/μl blood Lifespan in blood 7-10 days
30% sequestered in spleen (more if spleen enlarged)
Megakaryocyte development under influence of thrombopoietin and other growth factors

8. PLATELET ORGANELLES AND MEMBRANE COMPONENTS
Mitochondria
Glycogen granules (energy source)
Lysosomes
Dense granules (serotonin, Ca++, ATP, ADP)
Alpha granules (various plasma proteins, growth factors etc)
Membrane glycoproteins
GP Ib/IX/V: von Willebrand factor receptor (mediates platelet adhesion); 50K copies/platelet
GP IIb/IIIa (αIIbß3 integrin): fibrinogen receptor (mediates platelet aggregation; cryptic in resting platelets); 50-80K copies/platelet
Membrane phospholipids (inner leaflet)
Anionic/procoagulant
Arachidonic acid
“Flippase” keeps procoagulant molecules on inner leaflet – they are exposed with platelet activation

9. SOME IMPORTANT PLATELET MEMBRANE RECEPTORS
Thrombin
ADP receptor (several forms)
Thromboxane
Prostacyclin
P-selectin
Thrombopoietin (modulates TPO concentration in blood)
ß2-adrenergic receptor
Adenosine receptor
Serotonin receptor

10. PLATELET FUNCTION Maintain vessel integrity
Red cells leak from uninjured vessels if platelets absent (petechiae)
This effect mediated by various factors released by platelets, e.g., VEGF, angiopoietin, EGF
Help plug holes in damaged vessels
Adhesion (von Willebrand factor, GPIb)
Shape change
Activation
Release of granule contents
Aggregation (fibrinogen, GP IIb-IIIa)
Provide large membrane surface for thrombin generation, fibrin clot formation
Clot retraction

11. Single platelet filling a gap in the endothelium
Blood 2018;131:277

12. PLATELET ACTIVATORS AND INHIBITORS
Activators (partial list)
Collagen (in subendothelium)
ADP (from rbc, activated platelets)
Thromboxane A2 (from activated platelets)
Thrombin
Epinephrine
Inhibitors (partial list)
Nitric oxide (from endothelium)
Prostacyclin (Prostaglandin I2 - from endothelium)
Aspirin (blocks thromboxane synthesis)
Clopidogrel (Plavix® - blocks ADP receptor)

13. Platelet activation and inhibition by prostaglandins

14. PLATELET ADHESION TO
AREA OF VESSEL INJURY

15. PLATELET SPREADING
Patel et al, Blood 2003;101:929-36

16. PLATELET AGGREGATION

17. VON WILLEBRAND FACTOR
Large multimeric protein made by endothelial cells (and megakaryocytes)
Released into blood and subendothelium
Secretion stimulated by stress, exercise, epinephrine, thrombin and vasopressin analog DDAVP
Mediates platelet adhesion via GP Ib receptor
Largest multimers most effective
very large multimers can cause excessive platelet agglutination; they are broken down by ADAMTS13
Immobilization and unfolding of VWF (collagen binding, shear stress) increases adhesiveness
Deficiency or dysfunction of VWF causes bleeding tendency (von Willebrand disease)
VWF in blood binds factor VIII and protects it from inactivation

18. VON WILLEBRAND FACTOR
Single very large molecules visualized by electron microscopy
Electrophoresis showing range of multimer sizes

19. Biosynthesis of VWF
NEJM 2016;375:2067

20. VWF Mediates Platelet Adhesion and Facilitates Platelet Activation
NEJM 2016;375:2067

21. COMPONENTS OF THE PLATELET RESPONSE

22. FIBRIN CLOT FORMATION
Tissue factor (in subendothelium and other tissue) triggers enzymatic cascade that results in formation of thrombin
Platelet membrane supports and catalyzes these enzymatic reactions
Thrombin converts fibrinogen to fibrin
Fibrin polymerizes, polymers are crosslinked to form stable clot

23. Injury Initiation Propagation Thrombin Fibrinogen Fibrin TF VIIa IX
IXa
VIIIa Xa Va
Thrombin PT X XI
XIa
VIII V

24. Plasma proteins involved in coagulation
Size (kD)
Concentration (mg/dl)
Type of protein
Function (after activation)
Fibrinogen (factor I)
340
300
Structural
Polymerizes to form clot
Prothrombin (factor II)
72.5 10
VKZ*
Activates fibrinogen, V, VIII, XIII, protein C, platelets, XI
Factor V
350 2
Helper
Supports activation of II by Xa
Factor VII 50
0.1
VKZ
Activates IX and X
Factor VIII
Supports activation of X by IXa
Factor IX 57 1
Activates X
Factor X 59
Activates II
Factor XI
160
0.5
Zymogen
Activates IX
Factor XIII
320 3
Crosslinks fibrin, other proteins
*VKZ = vitamin K-dependent zymogen

25. TISSUE FACTOR LARGE VESSEL MONOCYTE MONOCYTE + ENDOTOXIN SMALL VESSEL
Am J Pathol 1989; 134:
LARGE VESSEL
MONOCYTE
MONOCYTE + ENDOTOXIN
SMALL VESSEL
Tissue factor is a ubiquitous, membrane-associated lipoprotein that is expressed by most cells. It is not normally expressed by endothelial cells or leukocytes, although expression can be induced in these cells by inflammatory cytokines (above, right). There is high expression in the adventita of blood vessels (above, left), brain, skin and mucosal tissue (the “hemostatic envelope”).

26. γ-carboxy glutamyl residues (vitamin K-dependent)
Generation of thrombin. Each step in the coagulation cascade requires an enzyme (in this case factor Xa), a proenzyme substrate (in this case prothrombin), a “helper” protein (here it is factor Va), a phospholipid surface (eg, a platelet membrane) and calcium. The enzyme and its substrate proenzyme contain calcium-binding regions that are vitamin K-dependent.

27. Blood 2013;122:2773

28. VITAMIN K Fat-soluble vitamin present in many foods
Some also made by gut bacteria
Needed to create calcium-binding sites on several clotting factors (II, VII, IX, X) and anticoagulant proteins (protein C, S)
Deficiency can cause bleeding tendency
Warfarin (Coumadin®) is an antagonist of vitamin K, used as an anticoagulant

29. Initiation of coagulation
Tissue factor exposed to blood, binds VIIa
Small amounts of VIIa in normal plasma
TF/VIIa activates X and IX
IXa/VIIIa activate X
Xa/Va activate II to form thrombin
Initial activation of V probably by Xa
TF/VIIa complex quickly inhibited by Tissue Factor Pathway Inhibitor (TFPI)
Amount of thrombin generated as a result of these reactions may be insufficient for clot formation

30. Propagation (amplification) of coagulation
IIa activates XI, VIII, V (positive feedback)
Xa and IIa activate VII
XIa generates more IXa
IXa/VIIIa generate more Xa
Xa/Va generate more IIa
IIa converts fibrinogen to fibrin (and XIII to XIIIa)
Fibrin crosslinked by XIIIa

31. Injury Initiation Propagation Thrombin Fibrinogen Fibrin TF VIIa IX
IXa
VIIIa Xa Va
Thrombin PT X XI
XIa
VIII V

32. The “Contact System”
When plasma is exposed to a negatively charged surface, this proteolytic pathway is activated
3 components: high molecular weight kininogen, prekallikrein, and factor XII
The end product of contact activation is factor XIIa, a serine protease that can activate factor XI
This system is responsible for the fact that blood clots when exposed to glass and other foreign surfaces
Polyphosphate released from platelets supports activation
Complete deficiency of any of the contact factors does not cause bleeding, suggesting that this pathway has little if any role in physiologic coagulation
System may contribute to regulation of fibrinolysis, angiogenesis, and inflammatory response

33. FIBRIN FORMATION Fibrinogen Thrombin Fibrin monomer Fibrin polymer
Blood 2005;106:2944

34. FACTOR XIII

35. Clot formation in a mouse following vascular injury
Platelets (red)
Tissue factor (green)
Fibrin (blue)
Platelets + tissue factor (yellow)
Tissue factor + fibrin (turquoise)
Platelets + fibrin (magenta)
Platelets + fibrin + tissue factor (white)
Celi et al, J Thrombos Haemost 2003;1:60

36. Propagation Initiation IXa-VIIIa Tissue factor-VIIa APC TFPI Xa-Va
Fibrinogen
Tissue factor-VIIa
IXa-VIIIa
Xa-Va
Initiation
Propagation
Thrombin
Fibrin
AT3
APC
TFPI
Regulation by anticoagulants (black boxes). TFPI- tissue factor pathway inhibitor, AT3- antithrombin, APC- activated protein C. Red arrows indicate inhibitory activity.

37. ANTITHROMBOTIC PROPERTIES OF ENDOTHELIUM
Sequesters tissue factor from blood
Prostaglandin I2 inhibits platelet aggregation
Heparan sulfate catalyzes inhibition of thrombin by antithrombin
Thrombomodulin catalyzes activation of protein C by thrombin
Nitric oxide is a vasodilator and inhibits platelet aggregation
ADPase inhibits platelet aggregation
Tissue factor pathway inhibitor neutralizes TF/VIIa

38. Blood 2019;133:906

39. Antithrombin mechanism
(substrate) T

40. THE ANTITHROMBIN SYSTEM

41. THE PROTEIN C SYSTEM A negative feedback loop that degrades factors Va, VIIIa
VIIIi Va
P S
VIIIa
IIa
P C
APC
IIa TM
E C

42. Tissue factor pathway inhibitorXa
TFPI Xa X Xa TF
VIIa TF
VIIa Xa
TFPI
VIIa TF

43. FIBRINOLYSIS Proteolysis of clot and replacement by connective tissue
Conversion of plasminogen to plasmin by plasminogen activator starts the process
Plasminogen activation catalyzed by fibrin
Alpha 2 antiplasmin and plasminogen activator inhibitors regulate the process
Products of fibrinolysis can be measured in blood (fibrin split products, D-dimer)
Plasminogen activators are useful in treatment of some thrombotic conditions (MI, stroke, etc)
Excessive fibrinolysis can cause bleeding (eg, DIC)

44. FIBRINOLYSIS

45. Riley et al, Lab Med 2016

46. FIBRINOLYSIS
Intact fibrin clot
Fibrin clot exposed to plasmin

47. Extravascular fibrin endocytosed by macrophages after plasminolysis
Blood 2016;127:1085