1. Rafi Ahmed, MD Hematology Oncology Fellow
Hemostasis
Rafi Ahmed, MD
Hematology Oncology Fellow

2. Hemostasis Process of blood clot formation at site of vessel injury
Response must be
Quick
Localized
Carefully regulated

3. Phases of hemostasis Formation of platelet plug
Propagation of clotting process by coagulation cascade
Termination of clotting by antithombotic control mechanisms
Removal of clot by fibrinolysis

4. Platelets
Production
Produced in Bone Marrow by fragmentation of cytoplasm of megakaryocytes
Differentiation of stem cell to production of platelets is approximately 10 days
Thrombopoetin is regulator of platelet production which binds to C-MPL receptors on platelets
Lifespan is 7-10 days

5. Platelet Structure
Glycoproteins of the surface involved in platelet reactions of adhesion and aggregation
Interior with calcium, ADP, ATP, serotonin in granules.
Alpha granules with heparin antagonist, PDGF, Beta-thromboglobulin, fibrinogen, VWF, and clotting factors

6. Platelet Function Formation of mechanical plugs during vascular injury
Adhesion
Aggregation
Secretion
Procoagulant activity

8. The Coagulation Cascade Primary Hemostasis
Consists of:
Platelet plug formations
Vascular spasm
Capillary endothelial adhesions
Temporary Fix
Lasts hours

9. Primary Hemostasis Platelets adhere to exposed endothelial tissue
Subendothelial microfibrils bind to subendothelial VWF (released from endothelium) This binds to GPIb-IX complex on platelet membrane
Platelets now “roll” along surface until GPIa/IIa binds to collagen
Platelet activation achieved by GPIIb/IIIa exposure to fibrinogen (primary) as well as VWF leading to platelet aggregation
Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

11. GP IB/IIA
GP IIB/IIIA
Collagen Receptor

13. Primary Hemostasis Platelets adhere to exposed endothelial tissue
Subendothelial microfibrils bind to subendothelialVWF (released from endothelium) This binds to GPIb-IX complex on platelet membrane
Platelets now “roll” along surface until GPIa/IIa binds to collagen
Platelet activation achieved by GPIIb/IIIa exposure to fibrinogen (primary) as well as VWF leading to platelet aggregation
Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

14. Primary Hemostasis: Platelet release reaction and aggregation
Collagen exposure/thrombin action:
Secretion of platelet granules
ADP, serotonin, fibrinogen, lysosomal enzymes, B thromboglobulin, and heparin neutralizing factors
Activation of platelet prostaglandin synthesis
Arachidonate release leads to thromboxane A2
Thromboxane A2
Potentiates platelet aggregation
Vasoconstrictive activity
Procoagulant Activity
Phospholipid mediated reactions
Factors IXa, VIIIa, and X in the formation of factor Xa
Factors Xa, Va, and prothrombin II results in thrombin
Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

17. The Coagulation Cascade Secondary Hemostasis
Same time as platelets are aggregating
Initiation substances sequentially activate by proteolysis a cascade of circulating precursor proteins
Factors XII, XI, IX, VIII form the intrinsic pathway
VIIa-Tissue Factor complex form the extrinsic pathway
Both pathways leads to factor X activation
Final pathway is converting prothrombin to thrombin which converts fibrinogen to fibrin
Leads to firm and stable plugs

18. Secondary Hemostasis PTT PT
Image retrieved from:

21. Cofactor
Cofactor
Cofactor

22. Tissue Factor Cell bound glycoprotein
Principal initiator of coagulation in vivo
Mostly in extravascular location but gets exposed to blood when endothelial barrier is breached

23. Tissue Factor is a Co-Factor
TF + VII TF-VIIA (active enzyme) X Xa (common pathway) IX IXa (intrinsic)

24. TFPI- Tissue Factor Pathway Inhibitor

25. Secondary Hemostasis In Vivo TF binds to factor VII
Activates factor VII (VIIa)
VIIa-TF complex
Activates factor IX and X
Factor X leads to small amounts of thrombin
Thrombin
Activates cofactors V and VIII
Amplification involves VIII and IX
Continues to propagate factor Xa
Activated factor X
Converts prothrombin to thrombin
Thrombin converts fibrinogen to fibrin monomer
XIIIa stabilizes fibrin polymers
Secondary Hemostasis
Hoffbrand et al. Essential Haematology. Oxford, UK: Blackwell Publishing, 2004.

26. Cofactor
Cofactor
Cofactor

27. Coagulation Factor Inhibitors
Important effect of thrombin is limited to site of injury
TFPI :tissue factor pathway inhibitor
Protein C and S: inhibitors of coagulation cofactor V and VIII
Thrombin binds to endothelial surface receptor thrombomodulin
Activates vitamin K dependent serine protease protein C
Protein S binds protein C to the platelet surface
Anti Thrombin III

28. Protein C and Protein S Thrombomodulin
Cell bound Protein on Endothelium
Binds to Thrombin (IIa) and inactivates
Thrombomodulin/Thrombin Complex
Protein C-> Activated Protein C (enzyme)
Protein S is cofactor for APC that binds it to platelet surface
Inactivates cofactors VIIIa and Va

29. Antithrombin III Antithrombin
Weak Inhibitor of enzymes until in activated confirmation
1000X fold activated in presence of heparin
Heparan sulfate proteoglycan on endothelial surface

30. Coagulation Factor Inhibitors
Fibrinolysis
Generate Plasmin
Remove Fibrin
Fibrin
Cofactor for destruction

32. Fibrinolysis

33. Approach to Bleeding Patient
Abnormal Bleeding May Result From
Vascular disorders
Thrombocytopenia
Defective platelet function
Defective coagulation

34. Pattern of Bleeding Vascular and Platelet Disorders
Associated with bleeding from mucous membranes and into skin
Coagulation Disorders
Bleeding in soft tissue or joints

35. Patient History Bleeding History Medication Use Past bleeding
During surgical procedures
Mucosal vs hemarthrosis
Medication Use
Aspirin, clopidogrel, ticlopidine, warfarin

36. Inherited Vascular Disorders
Hereditary hemorrhagic telangiectasia
Autosomal dominant
Dilated microvascular swellings
Pulmonary AV malformations
Recurrent GI hemorrhage
Connective Tissue Disorders
Ehlers-Danlos
Hereditary collagen abnormalities
Defective platelet aggregation

37. Acquired Vascular Defects
Senile purpura
Atrophy of supporting tissues of cutaneous blood vessels on dorsal aspects of forearms and hands
Purpura due to infections
Vascular damage from immune complex formation
Henoch-Schonlein
In children, follows acute infection
IgA mediated vasculitis
Scurvy
Vitamin C deficiency, defective collagen
Steroid purpura
Defective vascular support

38. Thrombocytopenia Production Defects Increased Destruction
Bone Marrow Failure
Increased Destruction
ITP
HIT
Drug induced
TTP/HUS
DIC
Splenic Pooling

39. Hereditary Disorders of Platelets
Thrombasthenia (Glanzmann’s Disease)
Deficiency in glycoprotein IIb and IIIa
Platelets fail to aggregate
Bernard-Soulier Syndrome
Platelets larger than normal
Deficiency of glycoprotein Ib
Defective binding to vWF, adherence, and no aggregation in ristocetin
Variable thrombocytopenia
Storage Pool Diseases

40. Acquired Platelet Functional Disorders
Liver disease
Decrease in clotting factors
Quantitative and qualitative platelet disorders
Cirrhosis: sequestration, decreased TPO
Uremia
Ecchymosis, epistaxis, GI/GU bleeding
Cardiopulmonary Bypass
Platelet interaction with non-physiologic surfaces
Hypothermia, complement activation, cytokines, thrombin
Adhesive properties altered

41. Von Willebrands Disease
VWF Functions
Primary Hemostasis
Platelet Adhesion
Platelet Aggregation
Secondary Hemostasis
Carrier of VIII in plasma (keeps it inactivated) until thrombin releases it
Synthesized in endothelial cells and megakaryocytes
Von Willebrand Disease
Autosomal dominant

42. Von Willebrand Disease
Mucocutaneous bleeding usually mild to moderate
Assays
VWF activity: measures VWF binding to plts
Ristocetin cofactor assay-gold standard
Collagen binding, VWF:activity assay
VWF antigen: concentration of protein (elisa)
Factor VIII activity (carrier function of VWF)
VWF Multimers, RIPA (Assays for classification)
Type 1: Partial quantitative deficiency of VWF
Type 2: (A, B, M, N) Qualitative defect in VWF
Type 3: Total deficiency of VWF

43. Quick Review of Hemostasis Screening Assays
Thrombin Time PT
PTT
Mixing Study

44. Thrombin Time
Simple test to measure conversion of fibrinogen to fibrin
Bovine thrombin added to citrated plasma, no calcium or phospholipid required (thus Lupus inhibitors don’t’ effect it)
Prolonged by
Heparin
Hypo or dysfibrinogenemia
High levels of FDP
Circulating Paraprotein
Antibody to bovine thrombin (cardiac patients)
Reptilase time- will exclude Heparin
Snake venom cleaves fibrinogen to fibrin
Not affected by AT III

45. Prothrombin Time (PT)
Thromboplastin (TF+PL+Ca2+) added to citrated plasma
Trigger Extrinsic Pathway
Usually insensitive to lupus inhibitors (since excess PL)
Usually insensitive to heparin (adding heparinase to reagent)
INR = (test PT/ mean normal PT) ^ISI
(International sensitivity Index)
Sensitivity to Deficiencies
Factors VII, X, V, II (30-50 u/dl) (60-160)
Fibrinogen mg/dL ( )

46. Activated Partial Thromboplastin Time APTT
Partial Thromboplastin= Phospholipid + Calcium
Activated: neg charged surface that activate contact factors (FXII, HMK)
Added to citrated Plasma with Calcium
Variable sensitivity to lupus inhibitors, heparin (less phospholipid)

47. Mixing Study
50-50 mix with normal plasma will correct if due to factor deficiency (50% of nl levels of any component factor)
If not correct then concern for inhibitor
Sometimes corrects but after incubation becomes prolonged- concern for inhibitor

48. Nl PT and prolonged PTT Disorders of intrinsic pathway Deficiencies
Factor VIII: Hemophilia A, VWD
Factor IX: Hemophilia B
Factor XI (variable bleeding history)
Factor XII, prekallikrein, HMWK (not associated with bleeding)
Inhibitors
Factor VIII (most common)
Factor IX, XI (rare)

49. Prolonged PT and normal PTT
Deficiency
Factor VII deficiency (range of phenotype)
Acquired inhibitors
Factor VII rare
Most commonly seen in warfarin therapy, early liver disease, vitamin K deficiency, DIC

50. Prolonged PT and PTT Deficiency of Factor X, V, II, I
Super therapeutic warfarin or heparin
Vitamin K deficiency, liver disease, DIC, fibrinolysis

51. And finally some anticoagulants
Vitamin K antagonists
Warfarin (factor 10, 9, 7, 2, C, S)
Activates Antithrombin III
Heparin, LMWH
Fondaparinux (pentasacchride)
Direct Xa Inhibitors
Rivaroxaban, apixaban
Direct Thrombin Inhibitors
Argatroban, Dabigatran