1. Examination of hemostasis
Stanislav Matoušek

2. Hemostatic disorders
+ thrombosis and embolism (e.g. DVT or MI or stroke)
Virchow’s trias – Endothelium dysfunction, stasis, blood factors
Procoagulational changes in the blood = HYPERCOAGUABILITY DISORDERS
- excessive bleeding
hemorrhagic diatheses = bleeding states
//Endothelium and vessel wall – purpuras/vasculopathies
//Platelets – thrombocytopenias and -pathias (common)
//Plasmatic coagulation system – coagulopathies

3. Causes and signs of bleeding disorders
Lack or defect of platelets
Blood vessel disorder
Lack of plasmatic factors
Mostly bruises and bleeding to joints
Excessive bleeding
Mostly petechiae

4. Disorders of primary hemostasis
Platelets and vessels

5. - - - - Endothelium Bleeding Platelets activated + + + Vessel injury +
Platelet thrombus -
Exposition of procoagulation phospholip. (pf3)
Coagulation cascade
fibrin web -
thrombin +
Vasoconstriction in injured vessel -
adrenalin +
serotonin +
Bleeding
Vessel injury
myocytes & fibroblasts
PDGF
ADP +
prostacycline PGI2 -
membrane phospholipids
Arachidonic acid
eicosanoids
PGG2 PGH2
Collagen Exposal
Exposal of von Wilebrand’s factor
Desquamated epithelium
Platelet adhesion
Platelets activated
thromboxan A2 +
kys. arachidonová
endoperoxidy
PGG2 PGH2
membr. fosfolipidy
Platelet aggregation
PAF
granulocytes, baso-, macrophages +
von Wilebrand’s factor
Endothelium

6. Thrombocytopenia 1) Production decreased↓ 2) Consumption increased ↑
A) with increased activity of thrombin
B) imuno-thrombocytopenia
C) other
3) Combination of both mechanisms

7. Platelet count 200 – 400 x 103/µL (109/L) = 200 000 – 400 000 /µL
The risk of spontaneous bleeding is low if the number of platelets is > /µL and blood vessels and coagulation system are intact

8. I) Thrombocytopenia from insufficient production
Aplastic disorder
Congenital
Acquired
Bone marrow damaged by medicaments (chemotherapy), chemicals (benzene), radiation, infection (HIV), autoantibodies
Infiltration of bone marrow (carcinomas, lymphomas, leukemia)
Myelodysplastic syndrome, Myelofibrosis
Defect in megakaryocyte maturation
vit. B12 and folate deficiency
Bone marrow aspiration (less megakaryocytes in aplastic)

9. 2) Thrombocytopenia from increased consumption
A) Increased activity of thrombin
Disseminated intravascular coagulation – DIC
activation in infections/malignities (extracellular proteases)-chronic DIC
B) Imunothrombocytopenia (e.g. autoantibodies)
Idiopathic thrombocytopenic purpura- autoantibodies produced with unknown cause
Acute vs. chronic
Secondary ( SLE, lymphomas, HIV)
After medicaments ….
Heparin induced
C) Other
Hypersplenism, artificial valve
TTP = Thrombotic thrombocytopenic purpura
Activity of thrombin, clotting tests PT and aPTT

10. Thrombotic thromboplastic purpura
? Cause is probably damage to endothelium - release of von Willebrand factor
- decreased degradation of vWf by its protease (antibodies against the protease, congenital?)

11. Purpura

12. 3) Combination of low production and increased consumption
Liver cirrhosis

13. platelet dysfunction = thrombocytopathy
Congenital
Acquired
aggregation inhibitors therapy (aspirin, ticlopidin)
platelets covered by IgA or IgM (multiple myeloma)
Dextran (infusion, often given in bleeding)
Uremia (Kidney failure)
Essential Thrombocytosis (one of myeloproliferative diseases analogy of polycythemia vera)
Normal number of platelets, bleeding time ↑
Clinically: Generally, it is difficult to stop bleeding (e.g. during surgery),

14. Bleeding time (Duke)
Standardized puncture of the earlobe, or standardized incision to the forearm with cuff inflated to 40 Torr .
We measure the time until the bleeding stops
2-5 min (2-9 min in forearm)
Increased in platelet dysfunction and in vonWillebrand disease (but not in Hemophilia)

15. vonWillebrand disease
Missing or defective vonWillebrand factor
The factor is important in platelet adhesion (primary hemostasis) and serves also as a carrier for factor VIII (decrease of concentration of factor VIII leads to defect in coagulation)
Combined bleeding of petechial and hemophilic type
Congenital or acquired
Prevalence 1%, but generally mild symptoms (heterozygotes)
Prolonged bleending time (Duke) and aPTT

16. Vasculopathies / purpuras
congenital
e.g. Ehlers-Danlos syndrom (defect of collagen)
Acquired
scurvy (vitamin C deficiency)
glucocorticoid excess
Purpura senilis
Henoch-Schoenlein purpura (children after an upper respiratory infection xx DD DIC in meningococcal infection!)

17. Tourniquet test (Rumpel-Leede capillary-fragility test)
A blood pressure cuff is inflated to a point little bit above diastolic blood pressure during 5 min (10 min).
Positive, if more that 5 petechiae per square inch are found.
Vasculopathies, dengue fever diagnosis (more than 20 petechiae).

19. Scurvy

20. Secondary hemostasis disorders
Plasma coagulation system

21. only by collagen contact
Prekallikrein
Kallikrein +
Intrinsic pathway
Intrinsic activation
only by collagen contact
HMWK
Subendothelium
(contact with collagen surface)
XII
(Hageman) + XI +
XIIa
(Hageman)
XIa + IX
IXa +
VIII
von Willebrand factor
Extrinsic pathway
Exogenous activ.
by tissue factor
Ca++
VII
VIIa +
VIIIa
von Willebrand factor + { + + } X Xa +
Platelet phospholipides
III
Tissue thromboplastin
Platelet phospholipides (pf.3)
Ca++ II
prothrombin
Ca++
Platelet phospholipides }
IIa
thrombin + V Va +
XIII
(fibrin stab. f.)
Ca++ +
Platelet phospholipides (pf.3)
Ca++
XIIIa
(fibrin stab. f.) +
Fibrin web
Weak bonds I
fibrinogen +
Fibrin web
Covalent bonds +

22. (contact with collagen)
Intrinsic pathway
(contact with collagen)
XIIa
(Hageman) +
Extrinsic pathway
(tissue factor)
Anti Vit. K +
XIa
(PTA)
Anti Vit. K { + }
Hemophilia B
VIIa
(konvertin)
IXa
(Christmas)
Hemophilia A
Membrane phospholipides
III
(tissue tromboplastin)
Ca++
VIIIa
(AF)
Platelet factor 3
(platelet phospholipides)
Ca++ +
Anti Vit. K II
prothrombin
Ca++
Platelet phospholipides }
IIa
thrombin + }
Anti Vit. K Xa
(Stuart-Prower) Va
(accelerin)
Ca++ +
IIa
(thrombin)
Fibrin web +
XIIIa
(fibrin stab. f.)
fibrin monomer I
(fibrinogen) +

23. Queen Victoria of Britain
Carrier of Hemophilia
X-linked
Great- grandson
Tsar Nicolai II of Russia
Tsarevich Alexei
Sufferer of Hemophilia

25. Partial Tromboplastin Time
(activated)
Partial Tromboplastin Time
PTT,aPTT, APTT
Intrinsic pathway
(contact with collagen)
XIIa
(Hageman) +
Extrinsic pathway
(tissue factor) +
XIa
(PTA) { + }
VIIa
(konvertin)
IXa
(Christmas)
Prothrombin Time,
PT, (Quick)
Membrane phospholipides
III
(tissue tromboplastin)
Ca++
VIIIa
(AF)
Platelet factor 3
(platelet phospholipides)
Ca++ + } Xa
(Stuart-Prower) Va
(accelerin) +
IIa
(thrombin)
Fibrin web +
XIIIa
(fibrin stab. f.)
fibrin monomer I
(fibrinogen) +

26. (contact with collagen)
Intrinsic pathway
(contact with collagen)
XIIa
(Hageman) +
Extrinsic pathway
(tissue factor) +
XIa
(PTA) { + }
VIIa
(konvertin)
IXa
(Christmas)
Membrane phospholipides
III
(tissue tromboplastin)
Ca++
VIIIa
(AF)
Platelet factor 3
(platelet phospholipides)
Ca++ + } Xa
(Stuart-Prower) Va
(accelerin) +
IIa
(thrombin)
Thrombin time
Fibrin web +
XIIIa
(fibrin stab. f.)
fibrin monomer I
(fibrinogen) +

27. aPTT Citrate or oxalate plasma,
We add phospholipids, Ca++, caolin or other negatively charged surface
Time until clot formation (depends on the lab)
~ s
Monitoring of heparin therapy, intrinsic and common pathway, hemophilia

28. Prothrombin Time (Quick test)
Citrate or oxalate plasma,
We add phospholipids, Ca++, and tissue thromboplastin Time until clot formation (depends on the used thromboplastin)
~ s
INR = PTtest/ PTnormal (norm 0.8 – 1.2)
Monitoring of anti vitamin K anticoagulant therapy (warfarin), extrinsic and common pathway

29. Evaluation of clotting screen
aPPT
PT(Quick)
trombo-cytes
Bleedin time
Common Causes
Vessel disorder, f. XIII deficiency ↑
Thrombocytopathy ↓
thrombocytopenia
Heparin, deficiency of VIII, IX, XI, XII, HMWK, prekallikrein
Factor VII deficiency
Warfarin, vit. K deficiency, I, II, V,X
vonWillebrand disease
Liver disease, DIC, sepsis

30. Inhibition of coagulation

31. (contact with collagen)
Intrinsic pathway
(contact with collagen)
Antithrombin 3
XIIa
(Hageman) +
Antithrombin 3
Extrinsic pathway
(tissue factor) +
XIa
(PTA) { + + }
Antithrombin 3
VIIa
(konvertin)
IXa
(Christmas)
Protein C
Membrane phospholipides
III
(tissue tromboplastin)
Ca++
VIIIa
(AF)
Platelet factor 3
(platelet phospholipides)
Ca++ + }
Antithrombin 3 Xa
(Stuart-Prower)
Protein C Va
(accelerin) +
Antithrombin 3
IIa
(thrombin)
Fibrin web +
XIIIa
(fibrin stab. f.)
fibrin monomer I
(fibrinogen) +

32. Antitrombin 3 Protein C Protein C
Antithrombin 3
Antitrombin 3
Antithrombin 3
Protein C
Antithrombin 3
Protein C
Antithrombin 3

33. Antithrombin 3
Protein C
Antithrombin 3

34. Heparin Heparin Heparin Platelet factor 4 Non-active Antithrombin 3
IXa
(Christmas) Xa
(Stuart-Prower)
XIa
(PTA)
XIIa
(Hageman)
Platelet factor 4
Heparin
Non-active
Antithrombin 3
Antithrombin 3
Antithrombin 3
IIa
(trombin)

35. (contact with collagen)
Intrinsic pathway
(contact with collagen)
Antithrombin 3
XIIa
(Hageman) +
Antithrombin 3
Extrinsic pathway
(tissue factor) +
XIa
(PTA) { + + }
Antithrombin 3
VIIa
(konvertin)
IXa
(Christmas)
Protein C
Membrane phospholipides
III
(tissue tromboplastin)
Ca++
VIIIa
(AF)
Platelet factor 3
(platelet phospholipides)
Ca++ + }
Antithrombin 3 Xa
(Stuart-Prower)
Protein C Va
(accelerin) +
Antithrombin 3
IIa
(thrombin)
Fibrin web +
XIIIa
(fibrin stab. f.)
fibrin monomer I
(fibrinogen) +

36. Fibrinolysis =thrombolysis

37. Plasminogen Activator
Thrombolysis
Plasminogen
activator
Plasminogen Activator
Inhibitor of
Inactivates Inhibitor of Plasminogen Activator
Inhibitor of
Plasminogen Activator
Aktivátor
plazminogenu
Protein S
Protein C +
non-active
protein C
Protein S
Protein S
Protein C
Protein C
VIIIa
(AF) Va
(akcelerin)
protein C
activator +
IIa
(thrombin)
Thrombomodulin
Endothelium

38. Hypercoaguability (thrombophilic) disorders

40. Most common hypercoagubility disorders
Mutation of factor V – Leiden mutation / insensitivity to protein C
Defect in Antithrombin III
Defect in Protein C
Defect in Protein S