1. Clotting, Hemostasis, Anticoagulation
Doç.Dr.Jale Çoban

2. Topics 1- Hemostasis overview 2- Coagulation 3- Bleeding / thrombosis
4- Laboratory tests
5- Patient samples

3. Hemostasis The balance between clotting and bleeding Clot Formation
Clot Dissolution
The balance between clotting and bleeding
Components of Hemostasis:
Vasculature
Coagulation proteins
Platelets
Hemostasis is, simply put, the balance between clotting and bleeding. Three major pathways are involved in hemostasis, and they include the vasculature, coagulation proteins, and platelets. Patients bleed or thrombose due to abnormalities of one or any combination of these three factors.

4. Hemostasis („hemo”=blood; sta=„remain”) is the stoppage of bleeding, which is vitally important when blood vessels are damaged.
Following an injury to blood vessels several actions may help prevent blood loss, including:
Formation of a clot

5. PHASES OF THE HEMOSTATIC PROCESS
Initiation and formation of the platelet plug
Propagation of the clotting process by the coagulation cascade
Termination of clotting by antithrombotic control mechanisms
Removal of the clot by fibrinolysis

6. Local vasoconstriction
is due to local spasm of the smooth muscle (symp. reflex)
PLT comming in contact with exposed collagen release: serotonin, ADP, TXA2, which accelerate vasoconstriction and causes PLT to swell and become more sticky

7. Formation of a platelet plug
Formation of a platelet plug. When blood platelets adhere to collagen in the wall of a defective blood vessel, they release ADP and synthesize thromboxane A2. These substances increase the adhesiveness of the platelets, causing more platelets to aggregate at the site of injury.

8. The functional response of activated platelets involves four different processes:
Adhesion — the deposition of platelets on the subendothelial matrix
Aggregation — platelet-platelet cohesion
Secretion — the release of platelet granule proteins
Procoagulant activity — the enhancement of thrombin generation

9. This process is facilitated by a plasma glycoprotein, von Willebrand factor (vWF). Produced in both endothelial cells and platelets, vWF forms bridges between receptors on the surface of platelets and collagen fibers in the connective tissue.

10. Formation of platelet aggregate
Injured blood vessel releases ADP, which attracts platelets (PLT)
PLT comming in contact with exposed collagen release: serotonin, ADP, TXA2, which accelerate vasoconstriction and causes PLT to swell and become more sticky

11. Formation of blood clot
In the formation of the clot, an enzyme called thrombin converts fibrinogen into insoluble protein, fibrin
Fibrin aggregates to form a meshlike network at the site of vascular damage

13. CLOTTING CASCADE AND PROPAGATION OF THE CLOT
The clotting cascade is depicted as consisting of an intrinsic and extrinsic pathway .

15. Stage I: Formation of prothrombin activator
Extrinsic pathway:
1. When blood comes in contact with injured tissue – tissue thromboplastin (F III) interacts with (F VII), and Ca2+ activating (F X).
Ca2+
Christmas factor
Anti hemophilic factor
Stuart factor
Stage I: Formation of prothrombin activator

16. Stage I: Formation of prothrombin activator
Intrinsic pathway:
2. Exposed collagen activates (F XII). Activated F XII activates plasma enzyme – plasma thromboplastin antecedent (PTA; F XI, which in the presence of Ca 2+ activates (F IX). F IX interacts with (F VIII), Ca 2+ to form a complex that activates (F X).
Ca2+
Christmas factor
Anti hemophilic factor
Stuart factor
Stage I: Formation of prothrombin activator

17. Stage I: Formation of prothrombin activator
Ca2+
Christmas factor
Anti hemophilic factor
3. Common pathway:
Activated F X in the presence of Ca 2+ forms complexes with accelerin (F V) to form prothrombin activator
Stage I: Formation of prothrombin activator

18. Stage II: conversion of prothrombin to thrombin
Ca2+
Prothrombin – inactive precursor of enzyme thrombin
In the presence of prothrombin activator and Ca2+ prothrombin is converted to thrombin
Thrombin itself increases its own rate of formation (positive feedback mechanism)

19. Stage III: conversion of fibrinogen to fibrin
fibrin-stabilizing factor
Fibrinogen – plasma protein produced by the liver
Thrombin converts fibrinogen to fibrin
Thrombin also activates fibrin-stabilizing factor (F XIII), which in the presence of Ca2+, stabilizes the fibrin polymer through covalent bonding of fibrin monomers

20. Ca2+
Calcium ions
Are required for promotion and acceleration of almost all blood clotting reactions
Except: activation of XII and XI (intrinsic mechanism)

21. Fibrinolysis

22. Clot Dissolution Plasminogen Plasmin Fibrin soluble fragments
Plasmin is formed from plasminogen - enzyme called activator (e.g. enzymes from urine, tears, saliva or bacterial enzyme streptokinase)
Plasmin as an enzyme is involved in breaking down fibrin into soluble fragments (fibrinolysis)
t-PA
Plasminogen Plasmin
Fibrin soluble fragments

23. Hirudo medicinalis produce Hirudin that inhibits Thrombin
Anticoagulants
Hirudo medicinalis produce Hirudin that inhibits Thrombin

24. Anticoagulants
Although tissue breakdown and platelets destruction are normal events in the absence of trauma, intravascular clotting does not usually occur because:
the amounts of procoagulants released are very small
natural anticoagulants are present (Antithrombin III, Heparin, Antithromboplastin, Protein C and S, fibrin fibers)

25. Natural anticoagulants
Antithrombin III – inhibits factor X and thrombin
Heparin from basophils and mast cells potentiates effects of antithrombin III (together they inhibit IX, X, XI, XII and thrombin)
Antithromboplastin (inhibits „tissue factors” – tissue thromboplastins)
Protein C and S – activated by thrombin; degrade factor Va and VIIIa

26. Severe reduction in the number of PLTs - thrombocytopenia
this causes spontaneous bleeding as a reaction to minor trauma
in the skin - reddish- purple blotchy rash
it may result from:
decreased production (toxins, radiation, infection, leukemias)
increased destruction (autoimmune processes)
increased PLTs consumption (DIC)
Thrombocytopenia
Hemorrhagic spots (petechiae)

27. Hepatic failure Most of the clotting factors are formed in the liver
Subconjunctival hemorrhage

28. Disseminated intravascular coagulation (DIC)
Widespread coagulation  thrombosis in small blood vessels  increased fibrinolysis, and depletion of coagulating factors  generalized bleeding
It may result from:
bacterial infections (endothelial damage)
disseminated cancers (release of procoagulants)
complications of pregnancy
severe catabolic states
Disseminated cervical cancer metastases (PET imaging)

29. Hemophilia A (lack of F VIII) and B (lack of F IX) are transmitted genetically and affect only males. Females carry the gen but do not show symptoms.
Von Willebrand’s disease – loss of large component of fVIII

30. Hemophilia A (lack of F VIII; 85%)
Spontaneous or traumatic subcutaneous bleeding
Blood in the urine
Bleeding in the mouth, lips, tongue
Bleeding to the joints, CNS, gastrointestinal tract
Mild hemophilia after injection in buttock

31. Tests of coagulation

32. LABORATORY EVALUATION
PLATELET COUNT
BLEEDING TIME (BT)
PROTHROMBIN TIME (PTT)
PARTIAL THROMBOPLASTIN TIME (aPTT)
THROMBIN TIME (TT)

33. PLATELET COUNT NORMAL 150,000 - 400,000 CELLS/MM3
< 100, Thrombocytopenia
50, ,000 Mild thrombocytopenia
< 50, Sev Thrombocytopenia

34. Bleeding time
The blleding time is intennded to measure platele function, but it is neither a sensitive nor a spesific test.
Platelet counts < /uL, aspirin, other platelet inhibitory drugs can prolong the bleeding time
Tne bleeding time can be prolonged in von Willebrand disease and other hereditary plateled function disorders.

35. Bleeding time – procedure:
A trained healtcare professionall makes a small incision on the patient’s arm, and every 30 sec. Gently blots the blood with filtter paper to see if the bleeding has stopped.The filter paper must not touch the wound.Prior to making the cut, a blood pressure cuff is placed on the patient’s arm at 40 mmHg.
Referance İnterval :
1 - 9min.
This may vary from lab to lab, depending on how the test is measured

36. Whole blood clotting time
The time taken for blood to clot mainly reflects the time required for the generation of thrombin
The surface of the glass tube initiates the clotting process. This test is sensitive to the factors involved in the intrinsic pathway
The expected range for clotting time is 4-10 mins.

37. Whole blood clotting time – procedure:
Clean the tip of the finger with an alcohol
Prick the finger tip with an automatic lancet
Note the time when blood first appears on the skin
Touch the tube to the drop of blood
Break gently 1cm of the tube at the end of 2 min, and every 30 sec these after
When fibrin is formed between the two broken pieces of tube the coagulation or clotting time is noted

38. "Intrinsic" and "extrinsic" coagulation pathways
Prothrombin Time: 12 – 14 sec
Activated Partial Thromboplastin Time
N: sec

40. The intrinsic pathway is initiated by the exposure of blood to a negatively charged surface (such as celite, kaolin, or silica in the in vitro activated partial thromboplastin clotting time [aPTT]).
The extrinsic pathway is activated by tissue factor exposed at the site of injury or tissue factor-like material (thromboplastin, TPL in the in vitro prothrombin clotting time [PT]).

41. Protrombin Time (PTT)
The PT measures the clotting time from factor VII through fibrin formation(extrinsic and common pathways).
PT prolangations caused by:
1.factor deficiencies involoving
-fibrinogen
-factor II (protrombin)
-factor V
-factor VII
-factor X
2.therapeutic anticoagulans Warfarin (Coumadine)
3. Lupus anticoagulants or by spesific factor inhibitors against
Fibrinogen or factor II,V,VII or X

42. Protrombin Time (PTT)
PT reagents is called thromboplastin (phospholipid with tissue factor and Ca++). It is added to patient plasma, and the time until clot formation is measured in seconds
(Referans interval: sec).
Tissue factor activates the extrinsic pathway.Phospholipid and Ca++ are required cofactors in the coagulation cascade.
Citrate in the blue top tube prevents clotting by chelating Ca++
Prolonged PT:
-   a vitamin K deficiency (vitamin K is a co-factor in the synthesis of functional factors II prothrombin), VII, IX and X)
-    liver disease
- Warfarin therapy (Coumadin)

43. Monitoring Warfarin (coumadin)
Warfarin is monitored by the international normalized ratio (INR)
The usual therapeutical goal is an INR of 2-3.

44. International Normalised Ratio (INR)
INR is calculated from the PT and is intended to allow valid comparisons of results regardless of the type of PT reagent used among different laboratories:
INR=(patient PT / mean normal PT)ısı
International sensitivity index(ısı) is a measure of the sensitivity of a particular PT reagent. Different PT reagents have different sensitivities to factor deficiencies.

45. A patient’s prothrombin time (PT) test result expressed as a ratio to a normal population control which has been standardized (or normalized) for the potency to the thromboplastin used in the assay.
It is standardized using a World Health Organization (WHO) international reference thromboplastin preparation, and determined using the equation:
INR = R^ISI,
where R is the PT ratio obtained with the working thromboplastin.

46. International Normalised Ratio (INR)
Therapeutic interval: Therapeutic interval for oral anticoagulant therapy:
Application: Monitoring oral anticoagulant therapy (eg. Warfarin);
note that heparin will not prolong INR (heparinase is included within the INR reagent)!!!!!!!!!!!!! For heparin therapy we monitor aPTT and/or aPTT ratio
The result for the PT is expressed as a ratio (prothrombin clotting time for patient plasma divided by time for control plasma);
Correction factor (International Sensitivity Index) is applied to the prothrombin ratio and the result issued as INR.

47. Activated Partial Thromboplastin Time (aPTT)
aPTT measures the cloting time from the activation of factor XII,through the formation of fibrin clot (intrinsic and common pathways)
aPTT prolangations are caused by:
Either factor deficiencies (especially of factors VIII,IX,XI and/or XII)
Monitor therapeutic anticoagulan HEPARİN

48. Activated Partial Thromboplastin Time (aPTT)
PTT reagent(phospholipid with
an intrensic pathway activator
such as silica, kaolin...) and
Ca++ are added to patient
plasma, and the time until
clot formation is measured in
second.

49. Most common case of prolonged aPTT – heparin!!!
prolongation of both the APTT and PT suggests factor X, V, II or I (fibrinogen) deficiency, all of which are rare
aPTT is normal in factor VII deficiency (PT prolonged) and factor XIII deficiency
Most common case of prolonged aPTT – heparin!!!

50. Role of PT, PTT: Warfarin, Heparin Monitoring
Anticoagulant PT
aPTT
Heparin
Normal
Prolonged
Warfarin (Coumadin)

51. Mixing TEST + + + Np F %100 Mix F %50 Hp F %0 Hp aPTT 70 sn
Mix 32 sn

53. Thrombin time (TT) – norm: 14-15 sec
Prolonged TT:
Heparin (much more sensitive to heparin than aPTT)
Hypofibrinogenemia

54. Selected causes of abnormal coagulation tests
Partial Thromboplastin Time (aPTT)
Prothrombin Time (PT)
Thrombin Time (TT)
Bleeding Time (BT)
Factor deficiency (except VII)
VII, X, V, II, fibrinogen deficiency
Low or absent fibrinogen
Thrombocytopenia
Antibodies to clotting factors
Antibodies
Dysfibrinogenemia, hypofibrinogenemia
Von Willebrand’s disease
Heparin
Warfarin; Vit K defficiency (mild to severe)
Drugs (Aspirin, NSAIDs, high dose penicillins, etc.)
Excessive Warfarin
Excessive Heparin
Cirrhosis, Uremia, PLTs dysfunction

55. Anticoagulants Heparin Warfarin (Coumadin) Direct thrombin inhibitors
Antiplatelet agents
As anesthesiologists you are very unlikely to be administering anything other then unfractionated heparin. This is because heparin has a short half life and can be reversed with protamine. All other classes of anticoagulants also inhibit coagulation more or less well but are too long acting to be used safely in the OR. In the very distant future we will have anticoagulants that target key clotting factors that are pharmacologically much cleaner than heparin, do not require monitoring and can be reversed easily and reliably.