
HEMOSTASIS Secondary hemostasis
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.
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
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
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
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
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
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
COMPLEXES ON MEMBRANE Extrinsic pathway Intrinsic pathway Common pathway Fibrin formation
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
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
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
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
F-Xa, F-Va, PL, Ca++ (Prothrombinase Complex) Prothrombin Thrombin Fragment 1.2
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
FIBRINOGEN • 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
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
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
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
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
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
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)
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
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
INTERRELATIONSHIP OF COAGULATION, FIBRINOLYTIC, KININ, AND COMPLEMENT SYSTEMS
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
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
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
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=2.5-9.5 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)
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 23-35 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