1. HEMOSTASIS

2. HEMOSTASIS COMPONENTS
Vessel wall
Platelets
Coagulation enzymes
Fibrinolytic system
Control mechanisms, including inhibitors
Normal hemostasis in involves the complex interaction of the vessel wall, circulating proteins and biochemical mediators, cells, promoters and inhibitors.Activation of hemostasis usually begins with damage to the vessel wall, exposing the subendothelium. Conversely, the intact vessel wall helps to maintain fluidity of blood, not simply through being a passive container wall, but also by synthesizing chemicals and proteins that actively contribute to the process. When the vessel wall is damaged, platelets are at the forefront of defense by sticking to the damaged area. The clotting enzymes contribute by developing a fibrin mesh that holds the platelets in place. Control mechanisms come into play to limit hemostatic process to the are of injury. Otherwise the whole body would “clot up” at the slightest stimulus.
NOTE: Normal hemostasis involves the complex interaction of the vessel wall, circulating proteins and biochemical mediators, cells, promoters and inhibitors. Activation of hemostasis usually begins with damage to the vessel wall, exposing the subendothelium. Conversely, the intact vessel wall helps to maintain fluidity of blood, not simply through being a passive container wall, but also by synthesizing chemicals and proteins that actively contribute to the process. When the vessel wall is damaged, platelets are at the forefront of defense by sticking to the damaged area. The clotting enzymes contribute by developing a fibrin mesh that holds the platelets in place. Control mechanisms come into play to limit hemostatic process to the are of injury. Otherwise the whole body would “clot up” at the slightest stimulus.

3. Sequence of Changes With Vascular Injury
Injury to vessel wall (endothelium) with resultant exposure of subendothelium
Platelet adhesion mediated by HMW vWF
Simultaneous activation of clotting enzymes
Platelet aggregation via fibrinogen receptors on platelets
Anchoring of platelet plug by cross-linked fibrin
Let’s expand on the sequence of events that occur when a vessel wall is injured and define a few basic terms. Injury exposes the subendothelial collagen and the soluble substances that are normally between the subendothelium and collagen.
NOTE: Let’s expand on the sequence of events that occur when a vessel wall is injured and define a few basic terms. Injury exposes the subendothelial collagen and the soluble substances that are normally between the subendothelium and collagen.

4. Normal Hemostasis
Vessel Wall
Exposed collagen
vFW
large multimers
Contraction
Tissue TPL
Platelet
adhesion
Activation of
Coagulation
Platelet
Aggregation
Thrombin
Definitive
Hemostatic
Plug
1º Hemostatic
Plug
This slide gives a diagrammatic representation of the
whole process and the functional interrelationships.
We will now go on to dissect the various components
of this process in more detail.
This slide gives a diagrammatic representation of the
whole process and the functional interrelationships.
We will now go on to dissect the various components
of this process in more detail.
Limiting Reactions

5. Platelet Components RECEPTORS vWF Fibrinogen Clotting Factors
Canaliculus
Actin
RECEPTORS
Alpha granules
Dense Granules
vWF
Fibrinogen
Clotting Factors

6. Platelet Granules Alpha granules vWF Fibrinogen PF4
Beta thromboglobulin
PDGF
Dense granules (delta)
ADP
Serotonin

7. Platelet Role in Hemostasis
vFW binding sites- platelet adhesion
Fibrinogen binding sites- platelet aggregation
Multiple binding sites for coagulation
factors - enhances appropriate steric relationships
Production of multiple chemical mediators
Binding sites for chemical mediators
First, lets look more closely at the role of platelets.
They play multiple roles in the hemostatic process, and,
contrary to the view held 30 years ago, when they were
thought to a small part of the process, many would
now consider them to have a central role. We already
have alluded to this by indicating that the first step after
injury is platelet adhesion to the subendothelium. This is
mediated by subendothelial HMW vWF binding to
specific receptor sites on the platelet membrane. In, addition
there binding sites that play a role in virtually every step
of the process. Also. chemical mediators are
synthesized by internal organelles, and they contain
a contractile protein that is responsible for clot retraction.
The coagulation enzymes are present in relative low
concentrations in plasma. Binding of certain of these factors
to specific receptors on the surface of activated platelets
allows them to line up in appropriate steric configuration,
catalyzing the process and limiting the reactions to the area
of injury.
First, lets look more closely at the role of platelets. They play multiple roles in the hemostatic process, and, contrary to the view held 30 years ago, when they were thought to a small part of the process, many would now consider them to have a central role. We already have alluded to this by indicating that the first step after injury is platelet adhesion to the subendothelium. This is mediated by subendothelial HMW vWF binding to specific receptor sites on the platelet membrane. In, addition there binding sites that play a role in virtually every step of the process. Also. chemical mediators are synthesized by internal organelles, and they contain a contractile protein that is responsible for clot retraction. The coagulation enzymes are present in relative low concentrations in plasma. Binding of certain of these factors to specific receptors on the surface of activated platelets allows them to line up in appropriate steric configuration, catalyzing the process and limiting the reactions to the area of injury.

8. von Willebrands Factor
Synthesized in megakaryocytes and endothelial cells - approx. 230,000 M.W.
Macromolecular multimer plasma: M.W. 1 x X106. Plasma carrier of Factor VIII, stabilizes it
Large molecular forms:
a. Most effective in platelet adhesion
b. Predominate in endothelial cells and subendothelium

9. Coagulation Cascade TISSUE FACTOR FACTOR VII VIIa (ACTIVATED) ( XI?)
IX IXa
TFPI
VIII
FACTOR X Xa
V, Ca
FACTOR II IIa
FIBRINOGEN FIBRIN MONOMERS
FACTOR XIII
IN VITRO CROSS-LINKED FIBRIN
PT (STABLE FIBRIN CLOT)
APTT PT
PHOSPHOLIPID
APTT
CONTACT ACTIVATION
FXII, FXI, FLETCHER,
FITZGERALD FACTORS
IX IXa
INHIBITION OF
CLOTTING
VIII
THROMBOMODULIN
PROTEIN C
PLATELET
ACTIVATION

10. Coagulation Cascade- PT
TISSUE FACTOR
FACTOR VII VIIa (ACTIVATED)
( XI?)
IX IXa
TFPI
VIII
FACTOR X Xa
V, Ca
FACTOR II IIa
FIBRINOGEN FIBRIN MONOMERS
FACTOR XIII
IN VITRO CROSS-LINKED FIBRIN
PT (STABLE FIBRIN CLOT)
APTT PT
PHOSPHOLIPID
APTT
CONTACT ACTIVATOR
FXII, FXI, FLETCHER,
FITZGERALD FACTORS
IX IXa
INHIBITION OF
CLOTTING
VIII
PHOSPHOLIPID
THROMBOMODULIN
PROTEIN C
PLATELET
ACTIVATION
(ENDPOINT)

11. Coagulation Cascade- APTT
TISSUE FACTOR
FACTOR VII VIIa (ACTIVATED)
( XI?)
IX IXa
TFPI
VIII
FACTOR X Xa
V, Ca
FACTOR II IIa
FIBRINOGEN FIBRIN MONOMERS
FACTOR XIII
IN VITRO CROSS-LINKED FIBRIN
PT (STABLE FIBRIN CLOT)
APTT PT
PHOSPHOLIPID
APTT
CONTACT ACTIVATOR
FXII, FXI, FLETCHER,
FITZGERALD FACTORS
IX IXa
INHIBITION OF
CLOTTING
VIII
THROMBOMODULIN
PROTEIN C
PLATELET
ACTIVATION
(ENDPOINT)

12. Control Mechanisms Intact endothelial cells Circulating inhibitors
Chemical mediators
Membrane bound receptors
Synthesizes activators of fibrinolysis
Circulating inhibitors
Protein C “system”
Fibrinolytic system
Endothelial component
plasma protein component

13. Intact Endothelium Limits Hemostasis INHIBITS PLATELET ACTIVATION
INACTIVATES PAI
FIBRINOLYSIS
INACTIVATES Va + VIIIa
PLASMINOGEN PLASMIN NO
PGI2
ADPase
INHIBITS Xa + THROMBIN
PROTEIN S
PROTEIN C PROTEIN Ca
ATIII
PLASMINOGEN
ACTIVATORS (tPA)
(uPA)
THROMBIN +
HEPARAN
THROMBOMODULIN
Subendothelium
vFW multimers

14. Intact Endothelium Limits Hemostasis – Fibrinolysis
INHIBITS PLATELET ACTIVATION
FIBRINOLYSIS
INACTIVATES PAI
INACTIVATES Va + VIIIa
PLASMINOGEN PLASMIN NO
PGI2
ADPase
INHIBITS Xa + THROMBIN
PROTEIN S
PROTEIN C PROTEIN Ca
ATIII
PLASMINOGEN
ACTIVATORS (tPA)
(uPA)
THROMBIN +
HEPARAN
THROMBOMODULIN
Subendothelium
vFW multimers

15. Intact Endothelium Limits Hemostasis INHIBITS PLATELET ACTIVATION
“Protein C System”
INHIBITS PLATELET ACTIVATION
INACTIVATES PAI
FIBRINOLYSIS
INACTIVATES Va + VIIIa
PLASMINOGEN PLASMIN NO
PGI2
ADPase
INHIBITS Xa + THROMBIN
PROTEIN S
PROTEIN C PROTEIN Ca
ATIII
PLASMINOGEN
ACTIVATORS (tPA)
(uPA)
THROMBIN +
HEPARAN
THROMBOMODULIN
Subendothelium
vFW multimers

16. Intact Endothelium Limits Hemostasis - Chemical Mediators
INHIBITS PLATELET ACTIVATION
INACTIVATES PAI
FIBRINOLYSIS
INACTIVATES Va + VIIIa
PLASMINOGEN PLASMIN NO
PGI2
ADPase
INHIBITS Xa + THROMBIN
PROTEIN S
PROTEIN C PROTEIN Ca
ATIII
PLASMINOGEN
ACTIVATORS (tPA)
(uPA)
THROMBIN +
THROMBOMODULIN
HEPARAN
Subendothelium
vFW multimers

17. Production of Coagulation Factors
Synthesized in the liver-
All except Factor VIII
Vitamin K dependent
II, VII, IX, and X
Protein C, Protein S
Factor VIII- unknown

18. Vitamin K Dependent Enzymes: Factors II, VII, IX, X
Synthesized in liver
Serine proteases, inactivated by AT3
Activation on surface of biologic
membranes
Have an affinity for binding calcium

19. Coagulation Cascade- PT
TISSUE FACTOR
FACTOR VII VIIa (ACTIVATED)
( XI?)
IX IXa
TFPI
VIII
FACTOR X Xa
V, Ca
FACTOR II IIa
FIBRINOGEN FIBRIN MONOMERS
FACTOR XIII
IN VITRO CROSS-LINKED FIBRIN
PT (STABLE FIBRIN CLOT)
APTT PT
PHOSPHOLIPID
APTT
CONTACT ACTIVATOR
FXII, FXI, FLETCHER,
FITZGERALD FACTORS
IX IXa
INHIBITION OF
CLOTTING
VIII
PHOSPHOLIPID
THROMBOMODULIN
PROTEIN C
PLATELET
ACTIVATION
(ENDPOINT)

20. Coagulation Cascade- APTT
TISSUE FACTOR
FACTOR VII VIIa (ACTIVATED)
( XI?)
IX IXa
TFPI
VIII
FACTOR X Xa
V, Ca
FACTOR II IIa
FIBRINOGEN FIBRIN MONOMERS
FACTOR XIII
IN VITRO CROSS-LINKED FIBRIN
PT (STABLE FIBRIN CLOT)
APTT PT
PHOSPHOLIPID
APTT
CONTACT ACTIVATOR
FXII, FXI, FLETCHER,
FITZGERALD FACTORS
IX IXa
INHIBITION OF
CLOTTING
VIII
THROMBOMODULIN
PROTEIN C
PLATELET
ACTIVATION
(ENDPOINT)

21. Prothrombin Time Poor reproducibility from lab to lab in US
No good assayed standards
Many manufacturers
Many chemically different reagents
Many different types of instruments
Poor lot-to-lot reproducibility even from same manufacturer

22. Reporting Protime Results
Each laboratory must establish it’s own normal range using the instrument and reagents that it is using
It may have to be redone with each new lot of reagents, certainly, at least rechecked and verified- insist on it from the laboratory you use
Results should be expressed in seconds, not INR
Results should be compared to the normal range. The true “Control” value is meaningless for clinical use.

23. Prothrombin Time
“The INR is the answer to our prayers- Hallelujah”- NOT!!
Poor calibration by reagent manufacturers is the weak link in the chain
Intended only for inter laboratory comparisons in patients who are on steady state anticoagulation with coumadin: at least two weeks of therapy, ambulatory, normal activity and diet
Widely misapplied to express protime results in all other situations

24. Bleeding Time
Widely misused as a screening test for platelet function abnormalities
Can predict trends when used to study large populations
Cannot predict bleeding risk in individual patients - use for this purpose has been discredited

25. Screening for Hemostatic Defects
PT, APPT- sensitivity is too poor to pick up mild defects
Bleeding time- poorly reproducible, too many false positives and false negatives, Most common cause of a prolonged bleeding time- improperly performed
Best screen: good history

26. Screen for Platelet Abnormalities
No good tests, history
Immediate bleeding
Mucous membrane bleeding
Easy bruising
Petechiae

27. Screen for Clotting Factor Deficiencies
Delayed onset of bleeding
Large ecchymoses or hematomas
Bleeding into joints

28. Screening History for Bleeding Problems
Do you think you have a bleeding problem?
Does anyone in your family have a bleeding problem?
Easy bruising?
Previous hemostatic challenges:
Major surgery
Trauma
Extraction of impacted teeth

29. Bleeding Problems Pre-operative screening
Patient with suspicious history
Actively bleeding patient

30. Pre-operative Screening
Most common hereditary bleeding problems?
Acquired bleeding problems?
Sensitivity of screening tests?

31. Hereditary Bleeding Disorders
von Willebrand’s disease - platelet
function
Storage pool disease (delta granule deficiency) - platelet function
Factor VIII deficiency (Hemophilia A)
Factor IX deficiency (Christmas disease)
Factor XI deficiency

32. Patient with Suspicious History
Refer to laboratory or specialist that specializes in bleeding disorders.

33. Actively Bleeding Patient
Focal bleeding - catgut insufficiency
Generalized bleeding -
Thrombocytopenia
Vitamin K deficiencies - common
DIC - most over-diagnosed cause of bleeding in the acute care/ICU setting. Primary fibrinolysis - rare

34. Acquired Bleeding Problems
drug-induced platelet function defects
Thrombocytopenia
vitamin K deficiency
Liver disease
Coagulation inhibitors
Post viral
Misc. others
Idiopathic

35. Vitamin K Deficiency
Appropriate clinical setting: a. Poor or no oral intake b. Broad spectrum antibiotics
Prolonged PT, PTT with a normal platelet count and fibrinogen - presumptive diagnoses of Vitamin K deficiency

36. Acute DIC: A clinical-pathologic Dx
Severely, acutely ill patient (not clinically stable).
Decreasing platelet count and/or fibrinogen.

37. Acute DIC Principles
Most over-diagnosed cause of bleeding in a hospital/ICU setting.
Should be approached as a diagnosis of exclusion
If it is the only diagnosis you can think of, you are over your head. GET HELP
Vitamin K deficiency is much more common.
Many other factors are more likely to be the cause of thrombocytopenia.

38. Possible DIC Run all tests on the same specimen:
PT, PTT, Fibrinogen, FDP Platelets
(Factor V, Factor VIII).
It may take sequential testing to establish diagnosis.

39. Sources of Vitamin K Diet- Fresh, green leafy vegetables
Synthesis by bacteria in the intestinal track
Typical ICU/acutely ill, hospitalized patient
No or poor oral intake
Broad spectrum antibiotic therapy
Increased vitamin K requirement because of illness
Result: Acquired vitamin K deficiency within two to three days of admission

40. Vitamin K Dependent Factors
II, VII, IX, X
PT - II, VII, X
APTT - II, IX, X

41. Vitamin K Deficiency vs. Acute DICPT
Prolonged
N or prolonged
APT
Prolonged
N or prolonged
FDP
Normal
Usually elevated
Fibrinogen
Normal
N or decreased
*Platelets
Normal
Usually decreased

42. Elevated Levels of FDP Recent surgery Acute thromboembolic event
Renal failure
Hepatic failure
Acute myocardial infarction
DIC
TTP/HUS
Primary fibrinolysis

43. Suggested Approach to the Bleeding Hospitalized Patient
Draw PT, APTT, FDP, fibrinogen and platelet count on same specimen as a panel. Do not try to use values drawn at different times.
Immediately give Vitamin K
Redraw panel in 4-6 hours.
K deficiency should show some degree of correction of PT and APTT within this time period
DIC should manifest itself by a decreasing fibrinogen without any significant correction of PT< APTT

44. Diagnostic Approach to Thrombocytopenia
Good history; medication - don’t forget heparin; Acuteness of onset; Underlying diseases
Physical - splenomegaly
Examination of blood smear by an experienced individual; platelet morphology, Other hematologic abnormalities
Bone marrow examination - almost never helpful in the absence of other hematologic abnormalities