1. Primary and Secondary Hemostasis

2. HEMOSTASIS Hemostasis
The process by which the body stops bleeding upon injury and maintains blood in the fluid state in the vascular compartment
Process is rapid and localized

3. HEMOSTASIS The primary players in hemostasis include
Blood vessels
Platlets
Plasma proteins
Coagulation proteins – involved in clot formation
Fibrinolysis – involved in clot dissolution
Serine protease inhibitors
Other minor players include
Kinin system
Complement system

4. HEMOSTASIS Defects Blood Vessels Plasma Proteins Platlets
In blood vessels, platlets or serum proteins can be corrected by utilization of the other 2 players
In 2 of the 3 players results in pathologic bleeding
Blood Vessels
Plasma Proteins
Platlets

5. HEMOSTASIS Primary hemostasis
Hemostasis can be divided into two stages
Primary hemostasis
Response to vascular injury
Formation of the “platelet plug” adhering to the endothelial wall
Limits bleeding immediately
Secondary Hemostasis
Results in formation of a stable clot
Involves the enzymatic activation of coagulation proteins that function to produce fibrin as a reinforcement of the platelet plug
Gradually the stable plug will be dissolved by fibrinolysis

6. FORMATION OF A STABLE PLUG

7. VASCULAR SYSTEM
Smooth and continuous endothelial lining is designed to facilitate blood flow
Intact endothelial cells inhibit platelet adherence and blood coagulation
Injury to endothelial cells promotes localized clot formation
Vasoconstriction
Narrows the lumen of the vessel to minimize the loss of blood
Brings the hemostatic components of the blood (platelets and plasma proteins) into closer proximity to the vessel wall
Enhances contact activation of platlets
Von Willebrand factor
Collagen fibers
Platlet membrane glycoprotein Ib
Activated platlets enhance activation of coagulation proteins

8. PRIMARY HEMOSTASIS Platelets Primary platelet plug
Interact with injured vessel wall
Interact with each other
Produce the primary hemostatic plug
Primary platelet plug
Fragile
Can easily be dislodged from the vessel wall

9. PLATELETS Platelets Small, anucleated cytoplasmic fragments
Released from megakaryocytes in the BM
Megakaryocyte proliferation is stimulated by thrombopoietin (TPO)
Humoral factor
Produced primarily by liver, kidney, spleen, BM
Produced at a relatively constant rate
Normal platlet count is x 109/L
Survive 9-12 days
Nonviable or aged platelets removed by spleen & liver
2/3 of platelets circulate in the peripheral blood
1/3 are sequestered in the spleen
These 2 pools are in equilibrium and constantly exchanging
Spontaneous hemorrhaging occurs when platlet count gets below 10 x 109/L

10. PLATLETS

11. MATURE MAGAKARYOCYTE

12. PLATLET RELEASE

13. PLATLET FUNCTION Platlets function to
Provide negatively charged surface for factor X and prothrombin activation
Release substances that mediate vasoconstriction, platlet aggregation, coagulation, and vascular repair
Provide surface membrane proteins to attach to other platlets, bind collagen, and subendothelium

14. PLATELETS Are the primary defense against bleeding
Circulate in resting state
Have minimal interaction with other blood components or the vessel wall
Morphology of resting platelet is smooth, discoid
When stimulated by endothelial damage, platlets become activated and they
Become round and ‘sticky’
Build a hemostatic plug
Provide reaction surface for proteins that make fibrin
Aid in wound healing
Platlet activation and plug formation involves
Adhesion
Shape change
Secretion
Aggregation

15. FORMATION OF PRIMARY HEMOSTATIC PLUG

16. PLATELETS AND SECONDARY HEMOSTASIS
Primary platelet plug is
Unstable and easily dislodged
Secondary hemostasis
Fibrin formation stabilizes and reinforces the platelet plug
Proteins interact to form fibrin assemble on negatively charged membrane phospholipids of activated platelets
System mediated by many coagulation factors present in an inactive form in blood.
Factors are assigned Roman numerals, I through XIII
All are produced in the liver. The von Willebrand factor is also produced in endothelial cells and megakaryocytes.

17. SECONDARY HEMOSTASIS
Coagulation factors are divided into three categories based on hemostatic function
Substrate –fibrinogen (Factor I), which is the main substrate used to make fibrin
Co-factors – accelerate enzymatic reactions
Enzymes
Coagulation factors are also classified by physical properties
Contact proteins
Involved in earliest phases of clotting
Partially consumed during coagulation
Found in serum
Prothrombin Group
Vitamin-K Dependant Clotting Factors
Most are found in serum

18. SECONDARY HEMOSTASIS The cascade theory of blood coagulation
Some drugs prevent clotting by acting as antagonists to Vitamin K (Warfarin and Coumadin)
Fibrinogen Group
Thrombin-Sensitive Clotting Factors
All are acted upon by thrombin in the process of blood coagulation
None found in serum
The cascade theory of blood coagulation
Involves a series of biochemical reactions
Transforms circulating substances into an insoluble gel through conversion of fibrinogen to fibrin
Requires
Plasma proteins
Phospholipids
calcium

19. CASCADE THEORY OF COAGULATION
Each coagulation factor is converted to an active form by the preceeding factor in the cascade
Calcium participates in some of the reactions as a co-factor
The blood coagulation cascade occurs on cell surface membranes.
The membrane localizes the reaction to the site of injury

20. SECONDARY HEMOSTASIS
Three different complexes assemble on the phospholipid membrane
The pathways for the formation of these complexes are
Intrinsic
Extrinsic
Common -Both intrinsic and extrinsic pathways converge to share factors in the common pathway
Both intrinsic and extrinsic pathways require initiation
Intrinsic - all factors involved in clot formation are in the vascular compartment
Extrinsic- is initiated when a tissue factor not found in blood enters the vascular system

21. COMPLEXES ON MEMBRANE Extrinsic pathway Intrinsic pathway
Common pathway
Fibrin formation

22. EXTRINSIC PATHWAY

23. INTRINSIC PATHWAY

24. COMMON PATHWAY Intrinsic and extrinsic pathways
Converge on the common pathway
In the final steps thrombin converts fibrinogen to soluble fibrin and the fibrin monomers are crosslinked to form a stable fibrin polymer.

25. COMMON PATHWAY

26. COAGULATION CASCADE

27. INHIBITION OF COAGULATION
Antithrombin (AT) is a potent physiologic inhibitor of thrombin, and several other factors involved in coagulation
In the presence of heparin, the inactivation of thrombin by AT is significantly increased

28. INHIBITOR PATHWAY OF COAGULATION

29. SUMMARY OF PRIMARY AND SECONDARY HEMOSTASIS
Sequence after vessel injury
Vasoconstriction
Controlled by vessel smooth muscle; enhanced by chemicals secreted by platelets
Platelet adhesion
Adhesion to exposed subendothelial connective tissue
Platelet aggregation
Interaction and adhesion of platelets to one another to form initial plug at injury site

30. SUMMARY OF PRIMARY AND SECONDARY HEMOSTASIS
Sequence cont’d
Fibrin-platelet plug
Coagulation factors interact on platelet surface to produce fibrin; fibrin-platelet plug then forms at site of vessel injury
Fibrin stabilization
Fibrin clot must be stabilized by F-XIIIa

31. FIBRINOLYSIS Activation of coagulation also activates fibrin lysis
Fibrinolysis results in a gradual enzymatic cleavage of fibrin to soluble fragments
Due to the activity of plasmin which is responsible for degradation of fibrin
Limits the extent of the hemostatic process
Reestablishes normal blood flow

32. PLASMIN ACTION
FDP= fibrin degradation products

33. KININ AND COMPLEMENT SYSTEMS
The kinin system is also activated by both coagulation and fibrinolytic systems
The kinin system is important in inflammation, vascular permeability, and chemotaxis
The complement system is activated by plasmin

34. INTERRELATIONSHIP OF COAGULATION, FIBRINOLYTIC, KININ, AND COMPLEMENT SYSTEMS

35. HEMOSTATIC BALANCE
The regulation of hemostatic and fibrinolytic processes is dynamic
Balance between
Pro- and anti-hemostatic mediators
Pro- and anti-fibrinolytic mediators
Balance can be upset if any components are
Inadequate
Excessive
Development of thrombi
Excessive local or systemic activation of coagulation
Sustained bleeding
Excessive local or systemic fibrinolytic activity

36. HEMOSTATIC BALANCE When hemostasis is delayed
Either platelet disorder or a coagulation defect
Bleeding episode may be prolonged
Imbalance created between
An abnormally slow hemostatic rate
A normal rate of fibrinolysis
An inadequate fibrinolytic response
May retard lysis of a thrombus and even contribute to its extension

37. BALANCE OF CLOTTING AND FIBRINOLYSIS

38. DIAGNOSIS OF BLEEDING PROBLEMS
Questions to address:
Is a bleeding tendency present?
Is the condition familial or acquired?
Is the disorder one affecting
Primary hemostasis (platelet or blood vessel wall problems)
Secondary hemostasis (coagulation problems)
Is there another disorder present that could be the cause of or might exacerbate any bleeding tendency?
Principal Presentations of bleeding disorders
Easy bruising
Spontaneous bleeding from mucous membranes
Menorrhagia – excessive bleeding during menstruation
Excessive bleeding after trauma

39. LABORATORY EVALUATION OF HEMOSTASIS
Three different categories of disorders may be found
Vascular and platlet disorders
Coagulation factor deficiencies or specific inhibitors
Fibrinolytic disorders
Bleeding disorders present differently depending upon the causative problem
Platlet disorders present as petechiae and bleeding into mucous membranes because of failure to form the platlet plug
Patients with coagulation defects (includes those with hemophilia) may develop deep spreading hematomas and bleeding into the joints with evident hematuria because of failure to reinforce the platlet plug.

40. LABORATORY EVALUATION
Tests to differentiate between these include
Platlet count
Peripheral blood smear evaluation
Ivy bleeding time (N= min) or platlet function analyzer (PFA)
Prothrombin time (PT) – test contains thromboplastin and calcium chloride and measures measures the extrinsic and common pathways (Normal=11-13 sec)
Activated partial thromboplastin time (APTT) -contact activators and a platlet substitute and calcium chloride are added to measure the intrinsic and common pathways (Normal usually sec, may vary depending upon analyzer used, reagents used, and patient population)

41. LABORATORY EVALUATION
Thrombin time (TT) – add thrombin and measure the time required for thrombin to convert fibrinogen to fibrin (common pathway) (N=15-22 sec)
Mixing studies with PT and APTT abnormal results -patient plasma is mixed with normal plasma to distinguish between factor deficiencies and coagulation inhibitors
If assay is corrected – due to factor deficiency
If partially corrected or uncorrected – due to inhibitor
Coagulation factor assays
Assays for fibrin degradation products – evidence of fibrinolysis

42. INHERITED QUALITATIVE PLATELET DISORDERS
Defects in platelet-vessel wall interaction
Most common disorder is von Willebrand disease
Deficiency or defect in plasma VWF
Defects in platelet-platelet interaction
Defects of platelet secretion and signal transduction
Abnormalities of platelet granules
Defects in platlet coagulant activity

43. LAB TESTS IN DISORDERS OF PRIMARY HEMOSTASIS
Platlet count PT
APTT
Bleeding time
Vascular disorder
Normal
Normal or abnormal
Thrombocytopenia
Decreased
Abnormal
Platlet Dysfunction
Usually Normal
Normal or Abnormal

44. DRUGS THAT ALTER PLATELET FUNCTION
A variety of drugs alter platelet function
Some are used therapeutically for their antithrombotic activity
For others, abnormal platelet function is an unwanted side effect
Effect on platelet function
Defined by an abnormality of bleeding time or platelet aggregation
Aspirin
Inhibits platlet aggregation
Inhibits platlet secretion

45. DISORDERS OF SECONDARY HEMOSTASIS
Hereditary vs acquired
Quantitative vs qualitative deficiencies
Laboratory screening tests (PT, APTT)
Does not differentiate quantitative vs qualitative disorders
Qualitative abnormal proteins will
Prolong clotting test
Be recognized by immunologically-based procedures
Activity assays
Essential when screening for deficiencies

46. VON WILLEBRAND DISEASE
Inherited hemorrhagic disorder
Genetically and clinically heterogeneous
Caused by a deficiency/dysfunction of VWF
Most common hereditary bleeding disorder
VWF
Multimeric blood protein
Performs two major roles in hemostasis
Mediates adhesion of platelets to sites of vascular injury
Is a carrier protein for F-VIII
Inherited defects in VWF may
Interfere with biosynthetic processing or disrupt specific ligand binding sites
Cause bleeding by impairing either platelet adhesion or blood clotting

47. HEMOPHILIAS Hemophilia A Hemophilia B Hemophilia C
Factor VIII Deficiency
Antihemophilic Factor
X-linked recessive disorder
Most common type of hemophilia
Hemophilia B
Factor IX Deficiency
Christmas Factor (from family of first patients diagnosed with the disorder)
Hemophilia C
Factor XI Deficiency
Autosomal recessive disorder seen primarily in the Ashkenazi Jewish population
Symptoms range from mild to severe

48. HEMOPHILIA Insufficient generation of thrombin by
F-IXa/VIIIa complex through the intrinsic pathway of coagulation cascade
Bleeding severity complicated by excessive fibrinolysis
Clinical severity corresponds with level of factor activity
Severe hemophilia
Factor coagulant activity <1% of normal
Frequent spontaneous bleeding into joints and soft tissues
Prolonged bleeding with trauma or surgery

49. HEMOPHILIA Moderate hemophilia Mild hemophilia
Factor coagulant activity 1-5% of normal
Occasional spontaneous bleeding
Excessive bleeding with surgery or trauma
Mild hemophilia
Factor coagulant activity >5% of normal
Usually no spontaneous bleeding

50. HEMOPHILIA – CLINICAL PRESENTATION
Readily diagnosed
In severe disease and patients with prior family history
Diagnosis based on
Unusual bleeding symptoms early in life
Age of first bleeding varies with severity of disease
Family history
Physical exam
Laboratory evaluation

51. HEMOPHILIA – TREATMENT
Replacement of clotting factor to achieve hemostasis
Annual cost for patient with severe hemophilia
$20, ,000
Various products available
Plasma-derived low, intermediate and high purity products
Plasma-derived ultrapure products
Ultrapure recombinant products
Replacement products – benefits vs risks
Blood-born pathogens
Hepatitis A, B, C, G; HIV, Parvovirus B-19
Thrombotic complications with some F-IX concentrations
Development of alloantibody inhibitors
Neutralize coagulant effects of replacement therapy

52. COAGULATION SCREENING TESTS IN CONGENITAL DEFICIENCIES
Platlet count PT
APTT
PFA TT
Congenital Deficiency N
XIII, mild deficiency of any factor, plasminogen activator inhibitor-1, α2 anti-plasmin A
VII – (extrinsic pathway)
XII, XI, IX, VIII, prekallikrein, high molecular weight kininogen (intrinsic pathway – includes hemophilias)
X, V, II (common pathway)
Fibrinogen (last part of common pathway)
A or N
Von Willibrands