1. Hemostasis: stopping the flow of blood
Thrombocytes = Platelets
Chap 36

2. Thrombocytes = platelets
They need homeostasis of their hemostasis (cessation of bleeding). You want to stop bleeding, but not excessive clotting.
Too much clotting, get thrombus or embolis.
Too little, get hemorrhaging.

3. NORMAL HAEMOSTATIC BALANCE
Naturally occurring anticoagulants and good vascular flow
(normally dominant)‏
Pro-coagulants and platelets.
Procoagulants vs. anticoagulants
endogenous vs. exogenous
Thrombosis (inappropriate clotting)
Bleeding
Failure to clot
By the end of this class:
You should be able to name naturally occurring coagulants and anti-coagulants

4. You have thousands of micro-tears in your vasculature every day
You have thousands of micro-tears in your vasculature every day. Platelets clot them constantly.
They come from a stem cell, hemocytoblast, from the myeloid stem cell line. It comes from a megakaryocyte, which is a differentiated cell. It sheds off some cytoplasm and pinches off a piece of membrane to become a platelet. Granules inside activated platelets are released, activating more platelets (positive feedback). An activated platelet changes from disc to spiny shape, like a sea urchin. When all clotting factors are consumed and there is still bleeding, this is disseminated intravascular coagulation (DIC), usually what causes death during baby delivery. There is an ultimate negative feedback to stop it.

5. Platelets On average there are 150- 200,000 platelets/µL of blood.
Platelets = thrombocytes (thrombus=clot)‏
Life span 8-12 days
Contain intracellular granules (α and ) that contain coagulation factors and ADP.
Actin, myosin (important contractile proteins)‏
Activated platelets form temporary plug.
On average there are ,000 platelets/µL of blood.
Produced in the bone marrow from megakaryocytes.
Production stimulated by thrombopoietin from liver/kidney
Cell membrane has receptors for von W.F., other platelets
Alpha granules have fibrinogen, plasminogen
Delta granules- ADP, serotonin, histamine

6. Genesis of Blood Products
Pluripotent
Stem Cell
Myeloid Stem Cell
Lymphoid
CFU-T
CFU-B
CFU-Eosin
CFU-Bas
CFU-GM
CFU-MEG
BFU-E
T-Cell
B-Cell
eosinophil
basophil
neutrophil
platelets
monocyte
macrophage
erythrocyte
Genesis of Blood Products
hemocytoblast
CFU-L
CFU-M
Copyright © 2006 by Elsevier, Inc.

7. ADP in the granules is one of the things that activate more platelets
ADP in the granules is one of the things that activate more platelets. Other things that activate them are collagen from under the endothelial cells, smoking, high estrogen levels (from BCP). There are K platelets per µl of blood. Platelets also have actin and myosin. When the clot is made, the actin and myosin interact with each other, pulls the ends of the blood vessel together.
Thrombopoeitin is released in liver and kidney to stimulate production of platelets.

8. Main phases of hemostasis
Vasoconstriction: artery is cut, both ends constrict
Platelet plug: severed ends exposed to collagen, activates platelets, ADP released, pile on top of each other, into an unstable platelet plug.
Coagulation: fibrin polymers weave around each other to form clot
Clot retraction: pulling cut end together
Clot dissolution
Tissue repair

9. Hemostasis Prevents blood loss thru the walls of damaged blood vessels
Also establishes a framework for further tissue repairs
6 main phases:
Vasoconstriction
Platelet plug formation
Coagulation (clot formation)‏
Clot retraction
Clot dissolution (fibrinolysis)
Tissue repair
Phase 1 and 2 are considered to be reversible, non-committed steps in hemostasis.
They are safety measures.

10. VASCULAR PHASE
The smooth muscle constricts, less blood spills out. How much blood is lost depends on the radius of the blood vessel, so it is important to apply pressure to a cut. Some chemicals increase vasoconstriction, such as thromboxane A2 (released by damaged endothelium), prostaglandin 2 (PG2). Thromboxane A2 is released by activated platelets, and it also acts to further activate more platelets (Positive feedback). Cyclooxygenase (COX-1 ) blocks arachidonic acid, which blocks production of thromboxane A2. Aspirin blocks COX-1 and you lose vasoconstriction ability.

11. Blood vessel damage
Smooth muscle in blood vessel wall contracts (vascular spasm)
Blood vessel diameter decreases
Blood loss slows
Myogenic contraction of smooth muscle
Enhanced by release of thromboxane A2 (vasoconstrictor) and local myogenic spasm, local factors, and nervous reflexes
Narrows damaged vessels

12. Vascular Phase Blood Vessel Damage Smooth muscle in BV wall
Myogenic contraction of smooth muscle
Enhanced by release of thromboxane A2 (vasoconstrictor) and local myogenic spasm, local factors, and nervous reflexes
Narrows damaged vessels
Smooth muscle in BV wall
contracts – vascular spasm
BV diameter 
Blood loss slows
Thromboxane A2 is a prostaglandin made by damaged endothelial cells and is also found within platelets.
This phase lasts less than 1 minute.

13. PLATELET PLUG
Platelets are normally disc shaped, but activated ones change shape and become spiny all over, exposing the receptors on them. Von Willebrand factor helps the platelet receptors anchor to the torn blood vessel. Inside the platelet, the granules release their contents, including ADP, which is dumped into the plasma. ADP is the #1 signal to recruit more platelets. Calcium, serotonin, thromboxane A2 are also released. As the platelets are activated and sticky, they will stick to exposed collagen and to each other in a platelet plug. It is not permanently fixed in place, that’s the clotting phase. In this stage, the platelet plug is unstable, and BP could dislodge it.

14. 2. Platelet Phase
Platelets are normally repelled from the blood vessel endothelial lining
When trauma occurs, traumatized platelets lyse and release their granular contents ADP and thromboxane A2. Occurs within 15sec of the injury
Platelets attach to sticky endothelial cells, the basement membrane, and to exposed collagen fibers. The endothelial cells release Von Willebrand factor (vWF also is a carrier for VIII).
These cause other platelet membranes to become “sticky”, then release…. (positive feedback) more release, more platelets… (amplification effect)‏
ADP  the primary stimulus for platelet aggregation
Thromboxane A2  causes platelet aggregation and local vasoconstriction
Serotonin  causes local vasoconstriction
Calcium ions
Mounds of platelets at damage site forms a plug.
This is NOT a clot! The plug is a weak “thumb in the leaky dam” fix. Can dislodge and leak again. Still need to form a firm web of fibers within the plug
As platelets become activated, they change shape from smooth disks to tiny spheres with exposed receptors and this leads to the aggregation of platelets to the damage site.

16. Factors that are against clotting
What prevents platelets from becoming activated? Endothelial cells also release a different prostaglandin called prostacyclin and nitric oxide help repel the platelets from the endothelial cells, so they cannot activate properly. These are anticoagulants.

17. Factors that promote clotting
A tear in vessel, collagen fibers provide a place for Von Willebrand factors to allow platelets to bind.
Just one platelet activated, releases ADP Thromboxane A2,, serotonin, calcium. These help clotting.

18. Let’s go over the activators of platelets again
Collagen
Activated platelets— sounds like positive feed back, yes?
ADP released from damaged RBCs and activated platelets
Thromboxane A2
Epinephrine (stress)‏
Thrombin— Look for this again later! Positive feedback mechanism!
Smoking
Estrogen (BCP): don’t smoke and take these, or can get a blood clot!
Active platelets change morphology from tiny discs to spheres

19. Platelet Facts
Thromboxane in platelets is formed from fatty acid precursor, arachidonic acid with the enzyme cyclooxygenase (COX-1 enzyme.)‏
Aspirin is an irreversible COX enzyme inhibitor; blocks formation of thromboxane; platelets don’t stick and plug is less likely to form. So, aspirin reduces tendency of those platelets to plug, and thus reduce blood clotting tendency.
Often prescribe aspirin to those with greater tendency to clot, to reduce risk of heart attack and stroke, (baby aspirin)‏
Must wait till exposed platelets are removed, turnover time, approx 1 wk-10 days
Undergoing surgery? Why is scheduling important?

20. COAGULATION
Deals with a series of clotting factors numbered 1-13, but there are only 12 of them!
The end result is to have fibrin threads create a meshwork; within it are platelets and RBCs trapped in it. But RBCs have no part in the clotting process, they just got trapped there. They contribute to the clot, but don’t make it. The fibrin meshwork makes the clot.
Calcium and coenzymes help enzymes do their job more efficiently.
If an enzyme is prefixed with pro-, or ends in -ogen, it is in its inactive form.
Prothrombin  thrombin
Fibrinogen  fibrin

21. 3. Coagulation
It is an amplification pathway (cascade; positive feedback).
The end result is a clot (thrombus). A web of fibers (fibrin threads) which support and make the platelet plug strong.
Requires clotting factors such as:
Calcium ions.
12 different factors, some synthesized by the liver
Many are proenzymes or zymogens (inactive enzymes)
The synthesis of some of them requires Vitamin K (II, VII, IX, X)‏

22. Common Clotting Factor Names and Numbers (add a subscript “a” to each when each becomes activated.)‏
I …………………..…. Fibrinogen
II ……………..…….. Prothrombin
III …………...………. Tissue thromboplastin
IV ………………...…. Calcium
VI………………...…. Doesn’t exist
VIII …………….….... Antihemophilic Factor (composed of 2 fragments: the small VIIIc and larger von Willebrand Factor)‏
IX ………………….. Christmas Factor (Christmas hemophilia)‏
XI and XII………….Contact Factors (don’t req calcium)‏
XIII ……………...….. Fibrin stabilizing factor
Mnemonic for memorizing the first 7 clotting factors:
Future = fibrinogen
Physiologists = prothrombin
Take fun = tissue factor
Classes = calcium
5 -13 Years = factors 5-13
Future Physiologists take fun classes years.

23. 3. Coagulation Phase (Clot formation)‏
Begins 30 sec or more after vessel damage occurs
Involves a sequence of steps leading to the conversion of fibrinogen (a circulating plasma protein) to the insoluble protein fibrin.
A network of fibrin grows and covers the surface of the platelet plug. RBCs and additional platelets are trapped in this tangle, forming a blood clot (thrombus) that effectively seals the damaged vessel wall.

24. Clotting Pathways
Extrinsic and intrinsic pathways release clotting factors that activate other clotting factors…positive feedback. The two pathways converge on one common pathway that starts with PAS (prothrombin activated substance). The extrinsic pathway makes PAS faster, but not as much of it. The intrinsic pathway is has more enzymes involved, so it is slower to make PAS, but it makes a lot of it. You don’t need to memorize the intrinsic and extrinsic pathway, just know the common pathway.
The common pathway starts with PAS, and it activates prothrombin  thrombin  fibrinogen  fibrin
Factor 13 links the fibers together to stabilize the plug.

25. The extrinsic pathway is activated when there is tissue damage.
The intrinsic pathway is activated when there is blood or vessel damage.
You can’t have one or the other pathway exclusively. Both will get activated.

26. Clotting Pathways PAS Fibrin polymers
The extrinsic pathway is triggered by damage outside the bloodstream, ex. surrounding tissue and begins with the release of a tissue factor (lipoprotein).
The intrinsic pathway is triggered by damage inside the bloodstream itself (damaged blood elements and endothelial wall). Platelet factor (PF-3) is a phospholipid that is released
Charged surfaces, such as glass or plastic tubing can trigger this pathway.
Heart and lung bypass?
The extrinsic and intrinsic pathways begin simultaneously, and converge at the final common pathway
Extrinsic Path
PAS
Fibrin polymers
Intrinsic Path
Anti-phospholipid syndrome= Normally phospholipid binding proteins contain/bind to PF-3 and modulate intrinsic clotting pathway. In APS- an antibody binds to the phospholipid binding proteins and thus PF-3 is left un-sequestered and thus leads to hypercoagulation!
Final Common Path
The extrinsic pathway is faster, but produces less product (PAS)‏
The intrinsic pathway is slower (more steps), but yields more product (PAS)‏

27. Final Common Pathway slower (2-6 minutes) many components (cascade)‏
Rapid and explosive in nature (15 seconds)‏
slower (2-6 minutes) many components (cascade)‏
The subscript “a” after a factor number indicates “activated”
Final Common Pathway

28. Polymers, Fibrin Threads
Final Common Pathway
Prothrombin Activator Substance (PAS)‏
Prothrombin
Thrombin
Fibrinogen
Fibrin monomers
Fibrin Stabilizing Factor
It ten plus 5, 4 and 3 equals (twenty) TWO, then 1+13 equals glue!
Polymers, Fibrin Threads

29. What factors come together to make PAS? 10, 5, 4, 3
Mnemonic to remember this:
If = (twenty) TWO, then equals glue!
2 = prothrombin (PAS activates factor 2, which activates thrombin.
1 = fibrinogen
13 = fibrin stabilizing factor

30. What does fibrinogen look like?
Fibrinogen has long fibers with a protein cap. If bacteria cut off the cap, the exposed fibrin heads have a negative charge. Their tail ends have a positive charge. Therefore, cut fibrinogen fibers will suddenly line up, head to tail, in a long series of fibers, making an inefficient clot. The bacteria that can do this can cause a person to bleed out (DIC).

31. What does fibrinogen look like?
6 polypeptide chain with a “cap” on the ends
Thrombin cuts cap off and creates fibrin
Fibrin ends have negative charge and this allows them to align and polymerize
Factor 13 needs to covalently link them together to make a stable fibrin thread
Thrombin chops these ends off -
Fibrin monomer (single monomer)‏
Factor thirteen links fibrin monomers

32. 4. Clot Retraction
Once the fibrin meshwork has appeared, RBCs & platelets stick to the fibrin strands.
Clot retraction (30-60 minutes)‏
The platelets then contract and the clot undergoes clot retraction which:
Pulls the torn edges of the vessel closer together, reducing residual bleeding and stabilizing the injury site
Reduces the size of the injured area, making it easier for fibroblasts, smooth muscle cells, and endothelial cells to complete repairs.
Tissue is repaired at the same time as clot retraction.
Once tissue repair is done, you need to dissolve the clot. Petechiae are little red dots in skin, from oozing, not enough platelets, after all the micro tears in your vessels. You are using your clotting factors right now!

33. CLOT DISSOLUTION AND TISSUE REPAIR: Fibrinolysis
If a clot stays, it is on the inside of your blood vessel, so it interferes with the endothelium from releasing clot factors in the future. Need to remove the rough clot to leave a smooth endothelium.

34. 5. Fibrinolysis- Clot dissolution
Plasminogen = circulating globulin
Plasmin = THE CLOT BUSTER! proteolytic enzyme, similar to trypsin
digest fibrin threads, fibrinogen, and other clotting factors
Significance: removal of tiny little clots
Plasminogen activators:
Tissue Plasminogen Activator (TPA), sometimes called the “clot buster” But is it really?
Urokinase—found in urine
streptokinase—from bacteria, so could cause antibody production
thrombin: now we see negative feedback. It converts plasminogen to plasmin. If we control thrombin, can control clotting we don’t want. Also need heparin and antithrombin-3.
Thrombin negative feedback

35. Manipulating Hemostasis- less clotting
Important anticoagulants (preventing a clot from forming) drugs include:
Heparin—released by basophils and given therapeutically
High affinity for anti-thrombin III and makes it a more powerful thrombin inhibitor
Protamine removes heparin from circulation
Used in open heart surgery, when blood must pass through tubing
Calcium chelators (citrate, oxalate, EDTA, EGTA) used externally only in collection tubes
Aspirin
irreversible COX-1 enzyme inhibitor
This lowers platelet levels of thromboxane, a prostaglandin needed to make platelets “sticky”
COO-
Ca++
Heparin makes anit-thrombin iii a more powerful thrombininhibitor
if blood goes back into bloodstream, calcium citrate only used
other ones used if blood needs to be in liquid form for a long time

36. During heart surgery, you take the blood out of the patient through a tube, so you line the tubing with heparin so it does not clot. After surgery, have to remove heparin from patient. Have to add protamine (from fish), which competes with heparin, makes heparin less available to bind to antithrombin 3, making it less able to bind to thrombin, so patient does not clot as easily. Add protamine to a drop of the patient’s blood, see how long it takes to clot; if too long, add a little more protamine, to figure out the right dose to give.

37. Manipulating Hemostasis- less clotting
Vitamin K deficiency
Get in green vegs, grains, meats, made by bacteria in large intestine
Vitamin K is a fat soluble vitamin needed as coenzyme in hepatocytes to make factors II, VII, IX and X. Hepatocytes put these factors into circulating plasma.
Added issue: Need bile salts to allow fat absorption of Vitamin K; bile salts are a product of hepatocytes. Need good liver function to absorb Vitamin K from intestine
Fat absorption problems--bile duct blockage, prevents uptake of Vit K
Coumadin/Coumarin/Warfarin– a natural substance found in certain grasses
is a competitive inhibitor of vitamin K activity in hepatocyte; blocks production of clotting factors II, VII, IX and X.
These factors must be available in plasma for clotting to occur
What if patient receiving coumarin eats lots of greens???
They should decrease their consumption of green veggies.

38. Clotting Times: Testing for deficiencies
You do not need to know normal clotting times. It depends on the lot number of the reagents in your lab.
You need to know this:
Activated partial thromboplastin time test (aPTT Test)
Detects deficiency of Factor VIII
Used for heparin treated patients
“Prolonged time” = deficiency
Prothrombin time test (PT Test)
Detects deficiency of Factor VII
Used in coumarin patients

39. Clotting Times: Testing for deficiencies
For a patient on coumarin, take a ratio of what is normal for them. Their time divided by normal, should be within a normal range.
“Warfarin PeT has an APiTite for heparin”
PeT= PT time
APiTite= APT Intrinsic, heparin

40. Clotting Times:Testing for deficiencies
XII
Intrinsic
Extrinsic XI IX
VII
VIII
Prothrombin time test (PT Test)
detects deficiency of Factor VII
Used in coumarin patients
“Prolonged time” = deficiency
Activated partial thromboplastin time test (aPTT Test)
detects deficiency of Factor VIII
Used for heparin treated patients
“Prolonged time” = deficiency X
25-35 seconds
PT test used for coumadin therapy
aPPT test used when using protamine to reduce heparin levels (want clotting)‏
11-15 seconds V
Prothrombin (II)‏
Thrombin

41. What are those tests used for?
aPTT
Heparin anticoagulant therapy (the target aPTT is usually 1.5 to 2.5 times that of a "normal" control sample (referred to as the "aPTT Ratio").
Pre-surgery to check coagulation times
Any person with unexplained bleeding
Post-surgery, when adding protamine to remove heparin from patient PT
often used to check how well anti-coagulant tablets such as warfarin and phenindione are working.
The drug's effectiveness can be determined by how much it prolongs the PT (measured in seconds), or increases the INR (a standardized ratio of the patient's PT versus a normal sample).
Also used to check for a bleeding disorder, liver disease or vitamin K deficiency, or to ensure clotting ability before surgery.
Most laboratories report PT results that have been adjusted to the International Normalized Ratio (INR). Patients on anti-coagulant drugs usually have a target INR of 2.0 to 3.0 (i.e. a prothrombin time 2 to 3 times as long as in a normal patient, using standardized conditions).
Needs standardizing due to tissue factor III having labile biological activity.
Labtestsonline.org

42. Manipulating hemostasis to prevent thrombi & emboli
A thrombus is formed when platelets begin to stick to the wall of an intact blood vessel
Platelets are often attracted to plaques – where endothelial and smooth muscle cells contain lots of lipids.
What problems could a growing thrombus pose?
If the clot (thrombus) breaks off and begins to drift in the bloodstream, it is called an embolus.
What problems could an embolus cause?
Above: Normal artery
Below: Same artery with a large thrombus (arrow)‏

43. Let’s review some of your endogenous Anti-coagulants
Smoothness of the endothelial lining
Anti-thrombin III
Plasminogen
Fibrin fibers—soak up thrombin and contain it
Heparin naturally released by endothelial cells
Prostacyclin (a prostaglandin) promotes vasodilation and is released by normal, uninjured endothelial cells

44. PATHOLOGY OF PLATELETS
Thrombocytopenia (Too few platelets)
Hemophilia
Disseminated Intravascular Coagulation

45. Thrombocytopenia Idiopathic thrombocytopenia purpura (ITP)‏
Reduced platelet counts: thrombocytopenia (<100,000/mm3) tendency to bleed
Petechiae- small, purple blood leaks below skin
Purpura- 1 cm
Unknown cause, or possibly autoimmune (seen often after viral infection) IgA or IgG targets platelets for destruction. More common in young and females
May recover after some weeks, sometimes not.

46. Hemophilia-What symptoms do you suppose are characteristic of hemophiliacs?
Refers to several different hereditary bleeding disorders with similar signs/symptoms (can be acquired, too through autoimmune disorders)‏
Hemophilia A
Results from lack of Factor VIII. Most common type (83%)‏
X-linked- carrier vs. disease
Von Willebrand Disease (12p)‏
A plasma protein that carries factor VIII
Hemophilia B
Less common. Due to deficiency of Factor IX
Also X-linked
Bleeding in the joints (hemarthrosis)
Bleeding and bruising in the soft tissue and muscles
Bleeding in the mouth from a cut or bite or loss of a tooth
Nosebleeds for no obvious reason
Blood in the urine (from bleeding in the kidneys or bladder)
Blood in the stool (from bleeding in the intestines or stomach)

47. Royalty like to choose particular families to marry into, or to marry within their family. Don’t marry your sibling or cousin! You probably have 4-8 recessive mutations, bring two parents like that together, kid will have a problem. Hemophilia is a serious coagulation disorder, lose a baby tooth and die from bleeding. The male kids are kept indoors to protect them from injury, but become emotional damaged and physically weak.

48. Hemophilia Y chromosome is short, missing portion
X,X female
X,Y male
= gene for factor VIII is carried on the X chromosome
Y chromosome is short, missing portion
Most common in males, they carry only one allele
Females
hemophilic female: what’s her genotype?
hemophilic “carrier” female: what’s her genotype?
Females can be carriers, and they can get the disease.

49. If father has disease and mother does not, and they have two boys and two girls, both girls will be carriers; both boys don’t have the disease.
If the father has no disease and mother is a carrier, one boy and one girl will not have the disease, and the other girl will be a carrier, and the other boy will have the disease.
Females only use one X chromosome, they shut the other one down, so we get female carriers without the disease. A calico cat gets its color that way. The colored patches are the random sites where the father’s color manifests.

50. Carrier vs. Disease Phenotype

51. Disseminated Intravascular Coagulation
abnormal bleeding and clot formation
coagulation and clot lysis in uncontrolled manner—excessive bleeding and thrombi leading to ischemia
due to massive tissue damage (say from a crushing accident)
depletion of clotting factors and hemolysis from damage to red blood cells in microcirculation from fibrin fibers
child birth (HELLP)- hemolysis, elevated liver enzymes, and low platelet count. Often misdiagnosed complication in pregnant women.

52. DIC is when there is so much tissue damage that all clotting factors are used, but there is still bleeding, no clotting factors are left, and the person bleeds out. HELLP is DIC during childbirth; the worst complication of childbirth. HELLP = Hemolysis elevated liver enzymes and low platelet count. We can figure that out before birth if we do a blood test.