1. Steps in Hemostasis (order the steps and explain your logic)
Clot retraction – contraction of platelets via actin and myosin pull fibrin threads together strengthening clot
Vascular spasm – vasoconstriction of damaged vessel
Fibrinolysis – breakdown and removal of the fibrin meshwork of the clot
Coagulation – activation of plasma proteins that result in the formation of a clot
Platelet plug formation - aggregation of platelets at the site of vessel damage
What do you think are the most important aspects of hemostasis that you need to know and understand?

2. Vascular spasm – vasoconstriction of damaged vessel
Platelet plug formation - aggregation of platelets at the site of damage
Coagulation – activation of plasma proteins that result in clot formation
Clot retraction – contraction of platelets pulls fibrin threads together strengthening clot
Fibrinolysis – breakdown and removal of the fibrin meshwork of the clot

3. Hemostasis: stoppage of bleeding
Objective Hemostasis
Hemostasis: stoppage of bleeding
Mechanisms:
Vascular Spasm: constriction of the damaged blood vessel
Platelet Plug: platelets adhere to each other and to the wall of the injured vessel to form a temporary seal
Coagulation: blood is transformed into a semisolid mass that surrounds and strengthens the platelet plug

4. + + Primary Hemostasis: Vascular Spasm/Platelet Plug Blood flow
Blood vessel damage
Platelet Plug
(2-4 mins)
Vascular Spasm
Platelets release ADP Platelets adhere serotonin and to each other thromboxane A2 forming a plug
(dense granules) Serotonin, Thromboxane A2 ADP + +

5. Vascular spasm - vasoconstriction
Neurogenic reflex mediated by pain receptors
Endothelin - released by endothelial cells
Serotonin (released by platelets)
Thromboxane A2

6. + + Blood flow Platelet Plug Vascular Spasm Blood vessel damage
(2-4 mins)
Vascular Spasm
Platelets release ADP Platelets adhere serotonin and to each other thromboxane A2 forming a plug
(dense granules) Serotonin, Thromboxane A2 ADP + +

7. Platelet plug formation – upon exposure to extracellular matrix molecules
Adhesion – von Willibrand’s factor
Shape change - swelling
Release reaction- granules release contents
Aggregation- mediated by thromboxane A2 , ADP, and thrombin

8. The Structure of A Blood Vessel
Platelets adhere to exposed sub-endothelial collagen fibers and other ECM components
Anything that interferes with collagen synthesis (aging, vitamin C deficiency, corticosteroid use) can lead to bleeding

9. Platelet Adhesion depends on the presence of Von Willibrands Factor, made by platelets and endothelial cells

10. Most common inherited bleeding disorder
Normal platelet count, but defective adhesion
Resulting in prolonged bleeding time

11. Secondary Hemostasis: Coagulation
The goal of coagulation is to form a fibrin meshwork!
The formation of a blood clot takes place in three phases:
1. Formation of Prothrombin Activator (prothrombinase)
2. Conversion of Prothrombin to Thrombin
3. Polymerization of Fibrinogen to Fibrin
slower pathway (3-6 minutes) shorter pathway (about 15 seconds)
all necessary factors are injured cells external to the blood in the blood release tissue factor (TF) which shortens the process
Intrinsic Pathway
Extrinsic Pathway

13. Intrinsic Pathway Extrinsic Pathway
a combination of activated factor X, Ca2+, PF3 and activated factor V
Prothrombin (inactive)
Fibrin (active)
Prothrombin Activator Complex
Thrombin (active)
Fibrinogen (inactive)

14. Ca2+ intrinsic What ion is needed in both pathways?
When blood coagulates in a test tube, is the extrinsic or the intrinsic pathway activated?
Antithrombin III inhibits activated Factor X – which pathway is inhibited – intrinsic, extrinsic or both?
Ca2+
intrinsic
both pathways

15. Why are newborns given a shot of Vitamin K?
Factor
Common name(s) I
Fibrinogen - has the highest concentration of all the clotting factors * II
Prothrombin *
III
Tissue Factor, Tissue Thromboplastin - released from injured endothelial cells * V
Labile Factor, Proaccelerin
VII
Stable Factor, Proconvertin  )
VIII - sex linked
Hemophilia A factor,  *
IX - sex linked
Christmas Factor, Hemophilia B Factor, Plasma Thromboplastin Component * X
Stuart Prower Factor * XI
Hemophilia C factor, Plasma Thromboplastin Antecedent *
XII
Hageman Factor
XIII
Fibrin Stabilizing Factor, Laki-Lorand Factor
PK - clot!
Prekallekrein, Fletcher Factor - also acts on plasminogen to form plasmin;
HMWK
High Molecular Weight Kininogen, Fitzgerald Factor
Note: 1. Factors I, II, V, VII, VIII, IX, X, XI, XII, XIII are made by the liver
2. The synthesis of factors II, VII, IX and X requires vitamin K
* The ones you have to know
Why are newborns given a shot of Vitamin K?

16. 1. platelets contain actin and myosin
Clot Retraction
1. platelets contain actin and myosin
Question: What other cells contain actin and myosin? What are these proteins used for?
2. platelets trapped within the clot contract and pull the fibrin threads together
3. Results:
1. the clot is strengthened and condensed
2. serum is squeezed out
3. the edges of the wound are drawn together
What is the difference between serum and plasma ???????
Muscle; Contraction
Serum = plasma minus clotting factors

17. Vessel Healing
Endothelial cells, vascular smooth muscle cells and fibroblasts undergo mitosis to repair the wall of the vessel
Mitosis is stimulated by:
1. platelet derived growth factor (PDGF), produced by platelets
2. vascular endothelial growth factor (VEGF) produced by many cell types

18. Recombinant PDGF can be used to promote wound healing
                                                            
74 year old white male with sensory neuropathy and poor fitting footwear developed a non-healing ulcer for 15 months on his right lateral foot.
On his initial visit, saucerization of the ulcer was performed. Daily topical Regranex was applied
After 6 weeks, the ulcer was completely healed.

19. Fibrin Degradation Products Plasminogen
Fibrinolysis
Enzymatic degradation of fibrin removes the clot when healing has occurred
Plasmin
Fibrin Degradation Products
Plasminogen
Fibrin
Plasminogen activators include:
1. TPA released by endothelial cells
2. Activated thrombin and factor XII
3. Urokinase released by endothelial cells, kidney cells, epithelial cells and cancer cells
4. Cytofibrokinase, a tissue enzyme
Streptokinase, bacterial product

20. Preventing Unwanted Clots
Smooth, intact endothelium (Which pathway?)
Circulation (movement) of the blood
Chemical factors
a. Prostacyclin, NO
b. Vitamin E Quinone
Leg Massagers Help To Prevent Clots

21. Objective 11 Clot Control Factor That Limit the size of Clots
1. Circulation of Blood
2. Inhibition of Activated Clotting Factors
- adsorption of thrombin into the clot removes it from circulation
- liver products that limit coagulation:
antithrombin III
protein C
heparin
TFPI = tissue factor protein inhibitor

22. Agents Used Clinically To prevent Clotting
Aspirin (reduces thromboxane production)
Heparin Sulfate (activates antithrombin III)
Warfarin and Coumadin, vitamin K inhibitors (How do these work?)
Blood banks routinely add calcium chelators to blood. Calcium chelators remove calcium from solution. Why do they add these chelators?

23. The medicinal leech, Hirudo medicinalis, produces hirudin which encircles and inactivates thrombin
Hirudin based anticoagulant pharmaceutical products - Lepirudin (Refludan) , hirudin derived from Hansenula (Thrombexx, Extrauma) and Desirudin (Revasc/Iprivask)

24. Important factors in hemostasis and where they are produced
1. Vascular spasm
Factor Source
Endothelin endothelial cells
Serotonin platelets
Thromboxane A2 platelets
4. Fibrinolysis
Factor Source
Tissue plasminogen factor(TPA)-endothelial cells
Activated XII liver
Thrombin indirectly liver
Urokinase endothelial cells, kidney cells
2. Platelet plug formation
Factor Source
Von Willebrand factor platelets, endothelial cells
Thromboxane A2-platelets
ADP platelets
Collagen (in vessel wall) fibroblasts
5. Clot prevention
Factor Source
Prostacyclin endothelial cells
NO endothelial cells, hemoglobin
Vitamin E quinone
Antithrombin III liver
Protein C liver
Heparin mast cells, basophils
3. Coagulation
Factor Source *
Prothrombin activator complex*
Prothrombin*
Thrombin*
Fibrinogen*
Fibrin
Tissue factor damaged tissue cells
PF3 activated platelets
Calcium diet, bones
Vitamin K intestinal bacteria
* Source for clotting factors is liver

25. Objective 14 Disorders Related to Hemostasis
Thrombus: a clot that develops and persists in an unbroken vessel or chamber of the heart

26. Embolus
: an intravascular clot that is floating free in the plasma

27. Disseminated Intravascular Coagulation:
 a systemic thrombohemorrhagic disorder involving unregulated fibrin formation and accelerated fibrinolysis

28. Thrombocytopenia
A platelet count of <50,000 /mm3 blood which may cause spontaneous bleeding
Deficient coagulation factor VII (hemophilia A), IX (hemophilia B), factor (XI) or others
Hemophilia

29. Prothrombin Time (PT)
Measures clotting time of plasma in the presence of an optimal concentration of tissue factor
Measures the overall efficiency of the extrinsic clotting system and the common pathway
Increased PT time may indicate deficiency of prothrombin, fibrinogen, or factors V, VII, or X
Partial Thromboplastin Time (PTT)
Measures the overall efficiency of the intrinsic pathway and the common pathway
PTT is prolonged in deficiencies of factors V, VIII, IX, X, XI, XII, HMW kininogen, heparin, prekallikrein, prothrombin, fibrinogen

31. Objective 15 Blood Groups
Agglutinogen: a glyoprotein found on the surfaces of RBCs
- agglutinogens elicit immunologic responses, such as antibody formation, when introduced into a non-compatible host

32. 49 of 50 : 17-T04ABOBlood_TAB.jpg 49 of 50 : 17-T04ABOBlood_TAB.jpg
                                                                                                                                                                                                                                                                                                                                                                                                
Agglutinin: a glyoprotein antibody produced by plasma cells and found in body fluids
Agglutinins are produced by non-compatible hosts when they are exposed to foreign aglgutinogens
Agglutination: a clumping reaction that occurs when agglutinogens are linked to agglutinins
© 2006 Pearson Education, Inc., publishing as Benjamin Cummings
© 2006 Pearson Education, Inc., publishing as Benjamin Cummings

33. Human Blood Groups
1.  
ABO
10.  
Diego
19.   Kx 2.
MNS
11.
Cartwright (or Yt)
20.
Gerbich 3. P
12. Xg
21.
Cromer 4. Rh
13.
Scianna
22.
Knops 5.
Lutheran   
14.
Dombrock
23.
Indian 6.
Kell
15.
Colton
24. Ok
7. 
Lewis
16.
Landsteiner-Weiner  
25.
Raph 8.
Duffy
17.
Chido/Rodgers
26.
Auberger 9.
Kidd
18. Hh
27.
Sutter

34. Clinically Important Blood Groups - The ABO System and the Rh System
A. The ABO System

35. 49 of 50 : 17-T04ABOBlood_TAB.jpg
                                                                                                                                                                                                                                                                                                                                                                                                
© 2006 Pearson Education, Inc., publishing as Benjamin Cummings

36. Agglutinigens and agglutinins
ABO Blood Types
Agglutinigens and agglutinins
Blood typing

38. The Rh System
Landsteiner was back at it again,
along with Alexander Weiner….
Together, they discovered the
Rh factor
The Rh Systen includes several agglutinogens:
Include C,D,E,c,e
D is the most immunogenic even though C and E are somewhat immunogenic

39. It was called the Rh factor because it was first found on blood cells isolated from a Rhesus monkey …..

40. Rh positive individuals have C,D,E in some combination
Rh negative individuals have no C, D or E
Rh negative individuals will develop anti Rh agglutinin (usually anti-D) if exposed to Rh positive blood
Inject Rh + blood
Produce anti-D agglutinin

41. Let’s practice:

42. Objective 18 Hemolytic Anemia of the Newborn -HDN
(Erythroblastosis Fetalis)
A disorder that may arise when a mother lacks an agglutinogen that is present on the RBCs of her fetus. The usual cause is an Rh incompatibility.
Mother: Rh negative Father: Rh positive Fetus: Rh positive
The process:
Rh negative mother carrying an Rh positive fetus:
Fetal Rh+ cells pass into the mothers bloodstream as a result of bleeding either during the pregnancy or during the birth process

43. Note: normally fetal RBCs and maternal blood are separated by a layer of placental cells (trophoblast)

44. Mother’s plasma cells produce anti-D agglutinin
Anti-D agglutinin can diffuse through the placenta and into the fetus during subsequent pregnancies
The anti D agglutinin will attack the fetal cells, causing hemolysis

46. The severity of the symptoms will depend on the amount of agglutinin produced by the mother and the amount of time that the the fetus is exposed to it. Specific problems include:
1. Anemia, hypoxia (can lead to brain damage, death)
2. Hyperbilirubinemia
3. Kernitcterus
4. Hepatosplenomegaly
5. Fetal ascites/edema

47. kernicterus Fetal ascites Erythroblasts in fetal blood
Neonatal jaundice

48. Treatment includes in utero transfusions, early induction of labor and exchange transfusions, phototherapy after birth
Prevention involves the administration of Rhogam to Rh negative mothers

49. Hemolytic Transfusion Reactions:
Reasons for Mismatches:
Human Error
Weak agglutinogens missed in typing
Atypical groups