1. HEMOSTASIS
Secondary hemostasis

2. SECONDARY HEMOSTASIS Also called the fibrin forming coagulation system
The purpose is to reinforce the platlet plug
System mediated by many coagulation factors present in an inactive form in blood.
Factors are assigned Roman numerals, I through XIII
Assigned in order of discovery, not place in reaction sequence
Each one has one or more common names or synonyms
Roman numeral followed by letter “a” indicates the activated form of the factor
All are produced in the liver. The von Willebrand factor is also produced in endothelial cells and megakaryocytes.

3. SECONDARY HEMOSTASIS
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 (factors V and VIII, HK, S, and C)
Enzymes
Serine proteases in active form
Transaminase in active form
Coagulation factors are also classified by physical properties

4. SECONDARY HEMOSTASIS Contact proteins
Factors XII, and XI, Prekallikrein (PK), and Kininogen (HMWK)
Involved in earliest phases of clotting
Partially consumed during coagulation
Found in serum
Also involved in
Fibrinolysis, kinin formation, activation of complement, inflammation
Congenital deficiencies
Often asymptomatic, except XI deficiency which usually results in a mild bleeding disorder

5. SECONDARY HEMOSTASIS Prothrombin Group
Vitamin-K Dependant Clotting Factors
Factors-II, -VII, -IX, -X, Prt C and S (and Z)
All contain γ-carboxyglutamic acid
Critical for Ca++ binding properties
Need Ca++ to bind to phospholipid surface
All but Factor-II found in serum
Drugs that act as antagonists to Vitamin K (Warfarin and Coumadin) inhibit the Vitamin K dependent carboxylation of glutamic acid

6. SECONDARY HEMOSTASIS Fibrinogen Group
Thrombin-Sensitive Clotting Factors
Factors I (fibrinogen), -V, -VIII, and -XIII
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

7. 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.
Clotting factors bind to the phospholipid membrane surface and rearrange until a complex including enzyme, substrate, and cofactor is formed.
Subendothelial tissue exposed with vascular injury and platlet surface provide the critical membranes
The membrane localizes the reaction to the site of injury

8. 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

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

10. EXTRINSIC PATHWAY
The extrinsic pathway is initiated when there is an injury to a blood vessel wall
In the presence of the tissue factor released from the injured nonvascular tissue (factor III or thromboplastin) and calcium (factor IV), factor VII is activated to factor VIIa
Factor VIIa activates factor X to Xa
Factor VIIa can also activate factor IX in the intrinsic pathway to IXa

11. EXTRINSIC PATHWAY

12. Factor XII is activated to XIIa.
INTRINSIC PATHWAY
Is initiated following exposure to negatively charged foreign substances such as collagen, subendothelium, or phospholipids.
Factor XII is activated to XIIa.
XIIa then interacts with the contact factors, prekallikrein and kininogen, to activate Factor XI to XIa
XIa then activates Factor IX to IXa in the presence of Ca++
IXa participates along with co-factor VIII:C, Ca++, and PF3 (a source of phospholipids), to activate Factor X which leads to the generation of thrombin
IXa -factor VIIIa-phospholipid-Ca++ is called the tenase complex

13. INTRINSIC PATHWAY
The complex of IXa, VIIIa, X, PF3,and Ca++ assembles on the surface of activated platlets (supply the phospholipid).
The surface provides the protective environment for the enzymatic reactions to occur
In plasma, VIII circulates as a complex in association with C which has the procoagulant activity and von Willebrand factor (vWF) which functions as a carrier protein.
VIII requires enhancement by the generated enzyme thrombin to amplify its activity

14. INTRINSIC PATHWAY

15. COMMON PATHWAY Intrinsic and extrinsic pathways
Converge on the common pathway
Both pathways activate Factor-X to Xa
Xa in the presence of Factor V, Ca++ and phospholipid converts prothrombin (Factor II) to its active form thrombin (IIa)
Thrombin then feeds back to activate factors VIII and V, converts fibrinogen to soluble fibrin, and helps to stabilize the fibrin monomer by converting factor XIII to XIIIa.
XIIIa cross-links the fibrin monomers to form a stable fibrin polymer

16. (Prothrombinase Complex) Prothrombin Thrombin
F-Xa, F-Va, PL, Ca++
(Prothrombinase Complex)
Prothrombin Thrombin
Fragment 1.2

17. Fibrinogen Fibrin Cross-linked Fibrin F-XIII
Thrombin F-XIIIa
Fibrinogen Fibrin Cross-linked
Fibrin
F-XIII
FP-A
FP-B
Once generated, thrombin converts fibrinogen to fibrin and activates F-XIII

18. COMMON PATHWAY

19. 3 stages of conversion of fibrinogen to fibrin
Proteolysis
Thrombin cleavage of fibrinogen results in fibrin monomers
Polymerization
Spontaneous self-assembly into fibrin polymers
Stabilization
Introduction of covalent bonds into fibrin polymers by XIIIa

20. THROMBIN ACTION

21. COAGULATION PATHWAY

22. COAGULATION CASCADE

23. THROMBIN
Thrombin plays a central role in the bioregulation of hemostasis in both normal and pathologic conditions.
Thrombin mediated mechanisms in hemostasis include
Acts as a potent platlet aggregating and secretion agent.
Amplification of the coagulation mechanism by activating co-factors V, VIII, and factor XI.
Thrombin activation of V and VIII act as a positive feedback to amplify the generation of more thrombin
Cleavage of XIII to XIIIa for fibrin stabilization
Complexing with endothelial cell thrombomodulin to activate protein C
Activated protein C inactivates co-factors Va and VIIIa, slowing down thrombin formation
Stimulation of tissue repair

24. INHIBITION OF COAGULATION
Antithrombin is a potent physiologic inhibitor of thrombin, factors Xa, IXa, XIa, and XIIa, activated protein C, and kallikrein
In the presence of heparin, the inactivation of thrombin and Xa by AT is significantly increased

25. INHIBITOR PATHWAY OF COAGULATION

26. 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

27. 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

28. CELL BASED MODEL OF HEMOSTASIS
Specific cellular receptors for coagulation proteins promote hemostasis which occurs in three overlapping phases
Initiation – involves tissue-factor bearing cell and production of small amounts of thrombin
Amplification – involves platlet activation and sets the stage for large scale throbin production
Propagation – the activated platlet surface protects factor XI and results in an explosive burst of thrombin formation

29. CELL BASED MODEL OF HEMOSTASIS

30. FIBRINOLYSIS Activation of coagulation also activates fibrin lysis
Fibrinolysis results in a gradual enzymatic cleavage of fibrin to soluble fragments
Limits the extent of the hemostatic process
Reestablishes normal blood flow
Important component
Localized activation of plasminogen (PLG) to plasmin
Plasmin enzyme system (the fibrinolytic system)

31. FIBRINOLYSIS
Plasmin
Responsible for degradation of fibrin (or fibrinogen)
Distinct protein fragments produced
Fibrin degradation products (FDPs)
Sites of plasmin cleavage
Similar in fibrin and fibrinogen
Plasmin also destroys factors V, VIII and other coagulation factors

32. PLASMIN ACTION

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. LABORATORY EVALUATION OF HEMOSTASIS
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 may develop deep spreading hematomas and bleeding into the joints with evident hematuria because of failure to reinforce the platlet plug. P Pl

39. LABORATORY EVALUATION
Three different categories of disorders may be found
Vascular and platlet disorders
Coagulatioin factor deficiencies or specific inhibitors
Fibrinolytic disorders
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)

40. LABORATORY EVALUATION
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)
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