rainwater@mymelody.com || 1st semester, AY 2011-2012

7 Coagulation and Fibrinolysis

Key Points

Physiologic hemostasis consists of the plasma coagulation, fibrinolysis, and anticoagulation protein systems.

Physiologic hemostasis is initiated by factor VIIa and tissue factor.

Physiologic hemostasis is not fully represented by current assays to detect coagulation abnormalities such as aPTT and PT.

Current assays to assess coagulation protein abnormalities have good diagnostic power to recognize specific defects in coagulation proteins.

Acquired coagulation protein defects more commonly reflect general medical disorders than specific protein defects.

History of the Coagulation Cascade

1964 = Ratnoff and MacFarland; Davies proposed the coagulation cascade starting with factor 12 to fibrin formation; Ratnoff (1955) earlier published that factor 12 deficiency was not associated with bleeding

Mid 1970s = cofactors for factor 12 activation (prekallikrein; high molecular weight kininogen) were not associated with bleeding state

1977 = Osterud and Rappaport: factor 7a is able to activate factor 10

2003 = Boze: factor 7a and tissue factor complex can not directly activate factor 10 but has to go thru factor 9 activation

At present, it is believed that there is no intrinsic pathway(this occurs only in the test tube) and the initiator for physiologic hemostasis is factor 7a and tissue factor complex

Physiologic hemostasis has two parts:

Cellular component = platelets, endothelial cells, neutrophils and monocytes

Plasma proteins producing clot, dissolution of clot (fibrinolytic system) and the naturally occurring serine protease inhibitors that terminate activities of coagulation and fibrinolytic systems

Endothelium and Platelets

Constitutive anticoagulation of the endothelium: 1. Glycosaminoglycans that bind anti-

thrombin 2. Thrombomodulin = locus where protein C is

activated by low levels of thrombin 3. Ectonucleotidase (CD 39) = degrades ADP 4. Prostacyclin and nitric oxide= prevents

platelet activation 5. Bind plasminogen, tissue plasminogen

activator, urokinase= contribute to fibrinolysis

Events after vessel injury

Collagen exposed platelets adhere to site of injury vWF binds platelets to subendothelium activation of platelets degranulation of platelets platelets aggregate temporary plug

As soon as vessel is injured, the following events are observed:

1. tissue factor is upregulated 2. TF forms complex with factor 7a which

activates respectively: a. 9 (which in turn activates) b. 10

3. Factor 10a in the presence of factor 5a, activates factor 2(prothrombin) to factor 2a (thrombin), the major clotting enzyme

Coagulation Protein System

Two types: zymogens and cofactors Zymogens: phospholipid bound(Vit K dependent) and

surface dependent pro enzymes Vit K dependent factors undergo carboxylation of

their amino terminals before they bound to phosphoplipid and only then, become functional

Cofactors: receptors for coagulation proteins

Proteins of the Coagulation System Surface bound Vitamin K-dependent Co-factors

Factor 12 Factor 7 HMWKininogen Pre-kallikrein Factor 9 Factor 8

Factor 11 Factor 10 Factor 5 Factor 2 Fibrinogen Protein C Protein S

Physiologic Protein Assemblies

1. Tenase: Factor 9a, thrombin activated Factor 8 on phospholipid surfaces or cell membranes in an ordered structure with Factor 10 to activate Factor 10a.

2. Prothrombinase: assembly of Factor 10a and thrombin -activated Factor 5a on phospholipid membranes or cell membranes in an ordered structure with Factor 2 (prothrombin) to accelerate its activation to Factor 2a (thrombin).

rainwater@mymelody.com || 1st semester, AY 2011-2012

Formation of fibrin and the fibrinolytic system Fibrin:

Fibrinogen has central E domain and D terminals thrombin cleaves fibrinopeptides A and B in the E domain soluble fibrin monomer these assemble with end to end and side to side association to form a non-covalent fibrin polymer activated Factor 13 crosslinks them into an insoluble fibrin clot

Plasmin cleaved soluble fibrinogen or fibrin Plasmin cleaves alpha chains from the D terminals,

making fragment XFragment X is asymmetrically cleaved into Fragment D and Fragment Y Fragment Y is further cleaved by plasmin into Fragment D and E

Plasmin cleaved insoluble, cross-linked fibrin When insoluble, cross-linked fibrin is proteolyzed by

plasmin, the neo-epitope between the D domains is preserved and the liberated fragment consists of the D dimer together with an E domain

Anticoagulant System

Anticoagulation Protein Systems

Protein C and S Activated Protein C:

1. Decreases thrombin formation = inactivates factors 5a and 8a

2. Stimulates fibrinolysis = liberates tP activator upon binding with ECPR which activates PAR (protease activated receptor)

Protein S (co-factor) o Free and bound (free form is the receptor)

Plasma serine protease inhibitor system

Current hypothesis for the initiation of the Hemostatic System TF+F7a activates F9; F9a activates F10 (maybe

inhibited by TFPI); F10a converts prothrombin to thrombin which converts fibrinogen to fibrin; fibrin stimulates plasmin which is inhibited by thrombin-activatable fibrinolysis inhibitor to decrease lysis activity

Thrombin accelerates convertion of F11 to F11a which accelerates F10 to F10a which acts on prothrombin

Approach to patients with hemostatic problem 1. Rule out initially acquired coagulation protein

deficiency 2. Coagulation factor prolongation may be due to:

a. True protein deficiency b. Inhibitor(Ig or abnormal production of

heparin, fibrinectin, cryoglobulin) c. Abnormal coagulation protein d. Enhanced clearance (Ag-Ab complex)

Review History Primary Secondary Onset after trauma: Spontaneous Delayed Site: Skin, mucous Deep tissues Clinical Examples: Thrombocytopenia Factor deficiency Platelet defect Liver disease vWD; scurvy Acquired inhibitors

rainwater@mymelody.com || 1st semester, AY 2011-2012

Physiologic hemostasis vs. Clinical Assays

Dictum: physiologic hemostasis is initiated by upregulation of TF + Factor 7a; NO clinical assay can assess this event

PT and aPTT monitor late event of actual clot formation; neither tests represent physiologic hemostasis

PT = addition of excess TF creates a very unphysiologic change in the normal stoichiometric relationship of factors, while aPTT measures more proteins than necessary for physiologic hemostasis

Activated PTT Negatively charged surface + phospholipid + plasma

with 3.2 g% sodium citrate incubate Add calcium chloride Measure clotting time A study of the (supposedly) intrinsic factors and the

common pathway factors

PT Tissue thromboplastin(animal derived or

recombinant; tissue factor plus exogenous phospholipid) + plasma incubate

Add 30 mM calcium chloride Measure clotting time Evaluates extrinsic and common pathway

Thrombin time/Reptilase time Purified exogenous thrombin + plasma Measure clotting time Measures fibrinogen Prolonged in: hypofibrinogenemia; dysfibrogenemia

Interpretation of abnormal coagulation tests Any defect below a specific point will lead to an

abnormal result above the defect E.g.:

1. Inhibitor of factor 8: abn factor 11 assay 2. Deficient/abnormal fibrinogen: affect

results of all clotting tests

Abnormal aPTT alone Associated with bleeding: factors 8, 9, 11 defects Not associated with bleeding: factor 12, prekallikrein,

high molecular weight kininogen, lupus anticoagulant

Abnormal PT alone and combined abnormal aPTT and PT Abnormal PT alone: factor 7 defects Combined:

1. Medical conditions: anticoagulation, DIC, liver disease, vitamin K deficiency, massive transfusion

2. Rare: dysfibrinogenemias, factors 10, 5, 2 defects

Rare bleeding disorders (not picked by tests of hemostasis) In order of frequency: Factor 13 defects, alpha-2

antiplasmin defects, plasminogen activator inhibitor/ defects, alpha-1 antitrypsin (Pittsburg)

If your patient has an inhibitor: Do mixing studies using the specific factor inhibitor

assay or the effect on PT or PTT For e.g., inhibitor for factor 8

Mixing normal and patients plasma should have lower value in the factor assay of factor 8 than the calculated expected value for the dilution.

Using PT, mix normal plasma and patients plasma Normalization of value excludes inhibitor but

prolongation is suggestive of an inhibitor

rainwater@mymelody.com || 1st semester, AY 2011-2012

The acquired coagulation protein deficiencies are common than hereditary deficiencies

Hereditary deficiencies: Henrys, pp 737-742 In practice, consider in your patients the following

common acquired coagulation protein deficiencies: anticoagulation, DIC, liver disease, Vitamin K deficiency, massive transfusion effect and inhibitors

Disseminated intravascular coagulation Pathophysiology: activation of the coagulation and

fibrinolytic system simultaneous formation of thrombin and plasmin consumption of coagulation factors and inhibitors

Causes: sepsis, malignancy, OB complications, massive tissue necrosis

Two phenotypes: 1. Hyperfibrinolytic state: prolonged PT and

PTT; decreased fibrinogen and platelet counts.

2. Prothrombotic state: normal PT and PTT; mildly decreased platelets, normal or increased fibrinogen

Confirmatory: D-dimer D-dimer can be positive also in resolving large vessel

thrombosis and soft tissue hematomas

Liver Disease Serious liver diseases prolong PT and PTT Reasons: decreased synthesis(all Vitamin K-dependent

factors 2, 7, 9, 10, protein C and S, protein Z) Usually with dysfibrinogenemia Prekallikrein 1st protein to decrease in liver disease Fibrinogen last protein to decrease in liver disease

Vitamin K deficiency After 4-6 weeks of antibiotics and parenteral nutrition

in the very ill patient Also seen in anatomic bypass of the small intestines,

malabsorption, biliary tract obstruction, warfarin, alcoholics

Massive transfusion Replacement of >1.5 blood volume in 24 hours Bleeding due to dilution of clotting factors, DIC or

acquired platelet dysfunction Lab DX: prolonged PT and PTT, decreased fibrinogen,

decreased platelets In a 70kgs patient given 1.5x his blood volume, the

circulating anticoagulant can be 735 ml

Acquired Coagulation Protein Inhibitors 1. Factor 8 inhibitor = most common; prolonged aPTT;

usually seen in the elderly with B cell malignancy, connective tissue disease, post=partum

2. Amyloidosis = with decreased factor 10 and 9 as a result of absorption of coagulation proteins onto amyloid protein

3. Hypergammaglobulinemia = with pan-inhibitors( multiple myeloma and Waldenstroms macroglobulinemia) and usually with dysfibrinogenemia

4. Lupus anticoagulant or Phospholipid antibody (thrombotic) = antibodies directed to epitopes of proteins bound to phospholipids

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