HEMOSTASISPrimary and Secondary Hemostasis

HEMOSTASIS

Hemostasis The process by which the body stops bleeding

upon injury and maintains blood in the fluid state in the vascular compartment

Process is rapid and localized

HEMOSTASIS

The primary players in hemostasis include Blood vessels Platlets Plasma proteins

Coagulation proteins – involved in clot formation Fibrinolysis – involved in clot dissolution Serine protease inhibitors

Other minor players include Kinin system Complement system

HEMOSTASIS

Defects In blood vessels, platlets or serum proteins can be

corrected by utilization of the other 2 players In 2 of the 3 players results in pathologic bleeding

Blood Vessels

Plasma Proteins Platlets

HEMOSTASIS

Hemostasis can be divided into two stagesPrimary hemostasis

Response to vascular injury Formation of the “platelet plug” adhering to the endothelial

wall Limits bleeding immediately

Secondary Hemostasis Results in formation of a stable clot Involves the enzymatic activation of coagulation proteins that

function to produce fibrin as a reinforcement of the platelet plug

Gradually the stable plug will be dissolved by fibrinolysis

FORMATION OF A STABLE PLUG

VASCULAR SYSTEM

Smooth and continuous endothelial lining is designed to facilitate blood flow

Intact endothelial cells inhibit platelet adherence and blood coagulation

Injury to endothelial cells promotes localized clot formation Vasoconstriction

Narrows the lumen of the vessel to minimize the loss of blood Brings the hemostatic components of the blood (platelets and

plasma proteins) into closer proximity to the vessel wall Enhances contact activation of platlets

Von Willebrand factor Collagen fibers Platlet membrane glycoprotein Ib

Activated platlets enhance activation of coagulation proteins

PRIMARY HEMOSTASIS

Platelets Interact with injured vessel wall Interact with each other Produce the primary hemostatic plug

Primary platelet plug Fragile Can easily be dislodged from the vessel wall

PLATELETS Platelets

Small, anucleated cytoplasmic fragments Released from megakaryocytes in the BM Megakaryocyte proliferation is stimulated by thrombopoietin

(TPO) Humoral factor Produced primarily by liver, kidney, spleen, BM Produced at a relatively constant rate

Normal platlet count is 150-400 x 109/L Survive 9-12 days Nonviable or aged platelets removed by spleen & liver 2/3 of platelets circulate in the peripheral blood 1/3 are sequestered in the spleen

These 2 pools are in equilibrium and constantly exchanging Spontaneous hemorrhaging occurs when platlet count gets

below 10 x 109/L

PLATLETS

MATURE MAGAKARYOCYTE

PLATLET RELEASE

PLATLET FUNCTION

Platlets function to Provide negatively charged surface for factor X

and prothrombin activation Release substances that mediate

vasoconstriction, platlet aggregation, coagulation, and vascular repair

Provide surface membrane proteins to attach to other platlets, bind collagen, and subendothelium

PLATELETS

Are the primary defense against bleeding Circulate in resting state Have minimal interaction with other blood components or

the vessel wall Morphology of resting platelet is smooth, discoid When stimulated by endothelial damage, platlets become

activated and they Become round and ‘sticky’ Build a hemostatic plug Provide reaction surface for proteins that make fibrin Aid in wound healing

Platlet activation and plug formation involves Adhesion Shape change Secretion Aggregation

FORMATION OF PRIMARY HEMOSTATIC PLUG

PLATELETS AND SECONDARY HEMOSTASIS

Primary platelet plug is Unstable and easily dislodged

Secondary hemostasis Fibrin formation stabilizes and reinforces the platelet plug Proteins interact to form fibrin assemble on negatively

charged membrane phospholipids of activated platelets System mediated by many coagulation factors present in

an inactive form in blood. Factors are assigned Roman numerals, I through XIII

All are produced in the liver. The von Willebrand factor is also produced in endothelial cells and megakaryocytes.

SECONDARY HEMOSTASISCoagulation factors 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 Enzymes

Coagulation factors are also classified by physical propertiesContact proteins

Involved in earliest phases of clotting Partially consumed during coagulation Found in serum

Prothrombin Group Vitamin-K Dependant Clotting Factors Most are found in serum

SECONDARY HEMOSTASIS Some drugs prevent clotting by acting as

antagonists to Vitamin K (Warfarin and Coumadin) Fibrinogen Group

Thrombin-Sensitive Clotting Factors 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.

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

Common pathway

Intrinsic pathwayExtrinsic pathway

Fibrin formation

EXTRINSIC PATHWAY

INTRINSIC PATHWAY

COMMON PATHWAY

Intrinsic and extrinsic pathways Converge on the common pathway In the final steps thrombin converts fibrinogen to

soluble fibrin and the fibrin monomers are crosslinked to form a stable fibrin polymer.

COMMON PATHWAY

COAGULATION CASCADE

INHIBITION OF COAGULATION

Antithrombin (AT) is a potent physiologic inhibitor of thrombin, and several other factors involved in coagulation

In the presence of heparin, the inactivation of thrombin by AT is significantly increased

INHIBITOR PATHWAY OF COAGULATION

SUMMARY OF 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

SUMMARY OF 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

FIBRINOLYSIS

Activation of coagulation also activates fibrin lysis Fibrinolysis results in a gradual enzymatic cleavage of

fibrin to soluble fragments Due to the activity of plasmin which is responsible for

degradation of fibrin Limits the extent of the hemostatic process Reestablishes normal blood flow

PLASMIN ACTION

FDP= fibrin degradation products

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

BALANCE OF CLOTTING AND FIBRINOLYSIS

DIAGNOSIS OF BLEEDING PROBLEMS

Questions to address: Is a bleeding tendency present? Is the condition familial or acquired? Is the disorder one affecting

Primary hemostasis (platelet or blood vessel wall problems) Secondary hemostasis (coagulation problems)

Is there another disorder present that could be the cause of or might exacerbate any bleeding tendency?

Principal Presentations of bleeding disorders Easy bruising Spontaneous bleeding from mucous membranes Menorrhagia – excessive bleeding during menstruation Excessive bleeding after trauma

LABORATORY EVALUATION OF HEMOSTASIS

Three different categories of disorders may be found Vascular and platlet disorders Coagulation factor deficiencies or specific inhibitors Fibrinolytic disorders

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 (includes those with hemophilia) may develop deep spreading hematomas and bleeding into the joints with evident hematuria because of failure to reinforce the platlet plug.

LABORATORY EVALUATION

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)

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)

LABORATORY EVALUATION 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

INHERITED QUALITATIVE PLATELET DISORDERS

Defects in platelet-vessel wall interaction Most common disorder is von Willebrand disease

Deficiency or defect in plasma VWF Defects in platelet-platelet interaction Defects of platelet secretion and signal

transduction Abnormalities of platelet granules Defects in platlet coagulant activity

LAB TESTS IN DISORDERS OF PRIMARY HEMOSTASIS

Platlet count

PT APTT Bleeding time

Vascular disorder Normal Normal Normal Normal or abnormal

Thrombocytopenia

Decreased

Normal Normal Abnormal

Platlet Dysfunction

Usually Normal

Normal Normal Normal or Abnormal

DRUGS THAT ALTER PLATELET FUNCTION

A variety of drugs alter platelet function Some are used therapeutically for their antithrombotic

activity For others, abnormal platelet function is an unwanted

side effect Effect on platelet function

Defined by an abnormality of bleeding time or platelet aggregation

Aspirin Inhibits platlet aggregation Inhibits platlet secretion

Hereditary vs acquired Quantitative vs qualitative deficiencies

Laboratory screening tests (PT, APTT) Does not differentiate quantitative vs qualitative disorders

Qualitative abnormal proteins will Prolong clotting test Be recognized by immunologically-based procedures

Activity assays Essential when screening for deficiencies

DISORDERS OF SECONDARY HEMOSTASIS

Inherited hemorrhagic disorder Genetically and clinically heterogeneous Caused by a deficiency/dysfunction of VWF Most common hereditary bleeding disorder

VWF Multimeric blood protein Performs two major roles in hemostasis

Mediates adhesion of platelets to sites of vascular injury Is a carrier protein for F-VIII

Inherited defects in VWF may Interfere with biosynthetic processing or disrupt specific

ligand binding sites Cause bleeding by impairing either platelet adhesion or

blood clotting

VON WILLEBRAND DISEASE

Hemophilia A Factor VIII Deficiency

Antihemophilic Factor X-linked recessive disorder Most common type of hemophilia

Hemophilia B Factor IX Deficiency

Christmas Factor (from family of first patients diagnosed with the disorder)

X-linked recessive disorder Hemophilia C

Factor XI Deficiency Autosomal recessive disorder seen primarily in the

Ashkenazi Jewish population Symptoms range from mild to severe

HEMOPHILIAS

Insufficient generation of thrombin by F-IXa/VIIIa complex through the intrinsic pathway of

coagulation cascade Bleeding severity complicated by excessive fibrinolysis

Clinical severity corresponds with level of factor activity

Severe hemophilia Factor coagulant activity <1% of normal Frequent spontaneous bleeding into joints and soft

tissues Prolonged bleeding with trauma or surgery

HEMOPHILIA

Moderate hemophilia Factor coagulant activity 1-5% of normal Occasional spontaneous bleeding Excessive bleeding with surgery or trauma

Mild hemophilia Factor coagulant activity >5% of normal Usually no spontaneous bleeding Excessive bleeding with surgery or trauma

HEMOPHILIA

Readily diagnosed In severe disease and patients with prior family history

Diagnosis based on Unusual bleeding symptoms early in life Age of first bleeding varies with severity of disease Family history Physical exam Laboratory evaluation

HEMOPHILIA – CLINICAL PRESENTATION

Replacement of clotting factor to achieve hemostasis Annual cost for patient with severe hemophilia

$20,000-100,000 Various products available

Plasma-derived low, intermediate and high purity products Plasma-derived ultrapure products Ultrapure recombinant products

Replacement products – benefits vs risks Blood-born pathogens

Hepatitis A, B, C, G; HIV, Parvovirus B-19 Thrombotic complications with some F-IX concentrations Development of alloantibody inhibitors

Neutralize coagulant effects of replacement therapy

HEMOPHILIA – TREATMENT

COAGULATION SCREENING TESTS IN CONGENITAL DEFICIENCIES

Platlet count

PT APTT PFA TT Congenital Deficiency

N N N N N XIII, mild deficiency of any factor, plasminogen activator inhibitor-1, α2 anti-plasmin

N A N N N VII – (extrinsic pathway)

N N A N N XII, XI, IX, VIII, prekallikrein, high molecular weight kininogen (intrinsic pathway – includes hemophilias)

N A A N N X, V, II (common pathway)

N A A N A Fibrinogen (last part of common pathway)

N N A or N A or N N Von Willibrands