Physiology of coagulation

Friday, 28 September 2012

Hemostasis
Blood must be fluid Must coagulate (clot) at appropriate time
Rapid Localized Reversible Thrombosisinappropriate coagulation

Fibrinogen Fibrin

Natural heparin like molecules that inactivate thrombin

Thrombin

Thrombosis
Thrombomodulin

Anti-thrombotic
mechanisms
Fibrinolysis
Breakdown of fibrin Plasmin (Plasminogen)

When bound to thrombin 1. Activates Protein C

2. Inactivates clotting factors

tPA

Clotting or thrombosis
Rudolph Virchow

Components of haemostasis
COAGULATION PROTEINS

Endothelium

Platelets

Endothelium

PROCOAGULANT
Plasminogen activator inhibitors

ANTICOAGULANT
Plasminogen activators

Fibrinolytic inhibitors Protein C Activated Protein C

Thrombin/Thrombomodulin

Thrombin/Thrombomodulin Tissue Factor Prostacyclin inhibitors

Von Willibrand Factor

Tissue Factor

Pro-coagulant role of the damaged endothelium

Synthesises tissue factor when damaged
Acquires the tissue factor from macrophages Tissue factor, when bound to VIIa is the major activator of the extrinsic pathway

Major site for the synthesis and storage of vWF

Increased Platelet adhesion Carrier protein for Factor VIII

Inhibition of Fibrinolysis
Thrombin activated fibrinolytic inhibitor Plasminogen activator inhibitor

Platelets
Anucleate sub-cellular fragments Arise from megakaryocytes in the marrow Normal count: 200-400 thousand/l Several surface receptors Activated by contact with extra-cellular matrix Aggregation to form a platelet plug Stabilisation by the formation of a fibrin clot

Platelets-2
Contact with collagen Swelling and pseudopod formation Contractile proteins contract forcefully Release of platelet granules Increased adhesion Adhere to collagen and vWF ADP and Thromboxane production Cascade of events lead to a platelet plug

Haemostasis
Primary
Vasoconstriction Platelet plug formation

Secondary
Coagulation Organisation of clot

www.homepage.montana.edu/~awmsg/Coagulation.ppt

www.homepage.montana.edu/~awmsg/Coagulation.ppt

Secondary haemostasis & Coagulation proteins

Fibrinogen

Fibrin

Fibrinogen

Thrombin

Fibrin

Factor 2

Produced in the Liver

Vit K dependant Post translational modification
Prothrombin (II) Xa Va

Thrombin (IIa)
Fibrinogen Fibrin

Extrinsic Pathway TF Prothrombin VIIa Xa Va

Thrombin
Fibrinogen Fibrin

Intrinsic pathway

XIIa
Extrinsic Pathway XIa TF Prothrombin IXa VIIIa VIIa Xa Va

Thrombin
Fibrinogen Fibrin

Intrinsic pathway

XIIa
Extrinsic Pathway XIa TF Prothrombin IXa VIIIa VIIa Xa Va

Thrombin
Fibrinogen Fibrin

Unstable clot

XIIIa

Stable clot

Fibrin

APTT/ACT Intrinsic pathway Prothrombin time

XIIa
Extrinsic Pathway XIa TF Prothrombin IXa VIIIa + Fibrinogen VIIa Xa Va +

Thrombin

Soft clot

Fibrin

XIIIa

Hard clot

Fibrin

Physiologic Inhibitors of coagulation

Antithrombin III Activated Protein C + protein S
Inactivates Va and VIIIa (via proteolysis)

Thrombomodulin (EC glycoprotein)

Binds to thrombin Decreases ability to produce fibrin Increases ability to activate Protein C

Role of vitamin K
Some clotting factors require a post-translational modification

before they are active in clotting

These factors are II, VII, IX, X, proteins C and S This PTM involves the addition of a COO- to certain Glutamate

residues in the clotting factors (Gamma-carboxyglutamate

residues) Essential for Ca2+ binding

This PTM requires vitamin K

Clot removal

Fibrinolysis

Fibrin

Plasmin

Fibrin degradation Products (FDP)

Fibrinolysis
Plasminogen

tPA

Fibrin

Plasmin

Fibrin Split Products (FSP)

Inhibitors of fibrinolysis

1. 2.

Plasminogen activator inhibitors (PAIs)

Tranexamic acid 2-antiplasmin (serpin)

Testing the hemostatic system

CBC Blood smear
Schistocytes and fragemented RBC- DIC Teardrop-shaped or nucleated RBC Myelophthisic disease Characteristic WBC morphologies seen in thrombocytopenia in infectious mononucleosis, folate, B12 deficiency, or leukemia

Testing the hemostatic system

Platelet count Thrombocytopenia : Less that 100,000/mL Spontaneous bleeding possible: Less than 20,000/mL Count does not have anything to do with functionality of platelet

Testing the hemostatic system

Bleeding time
Tests vascular integrity and platelet function Incision on volar aspect of the forearm 1mm deep and 1 cm long BP cuff inflated to 40 mmHg Normal < 8 minutes Borderline 8-10 minutes Abnormal 10 + minutes

Testing the hemostatic system

Bleeding time
Prolonged with platelet counts below 100,000 When prolonged with platelet count over 100,000 suggests platelet dysfunction Affected by Aspirin (permanent) and NSAIDs

Testing the hemostatic system

Prothrombin Time
Test of extrinsic and common pathways International Normalized Ratio used to compensate for differences in thromboplastin reagents Used for warfarin monitoring Elevated in patients with liver disease and abnormalities in vitamin K sensitive factors

Testing the hemostatic system

Partial Thromboplastin Time (PTT)
Tests intrinsic and common pathway Affected by heparin Can be effected by warfarin at supra-therapeutic levels due to effects on the common pathway

History and Physical

Platelet Disorders
More common in Women Petechiae, Purpura, mucosal bleeding
More commonly acquired

Coagulation Disorder
More common in Men
Delayed deep muscle bleeding, hemarthrosis, hematuria More commonly congenital

Other clotting studies

Fibrinogen level FDP D-Dimers Individual factor assays

BLEEDING DISORDERS

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?

DIAGNOSIS OF BLEEDING PROBLEMS

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

EASY BRUISING
Bruising with mild trauma Bruising without trauma
Petechiae - a Ecchymoses - b Hematoma - c

MUCOSAL BLEEDING & MENORRHAGIA

Epistaxis - nosebleed
History of recurrence

Gingival bleeding Hematuria, hemoptysis, hematemesis

Relatively uncommon presenting features

Menorrhagia

EXCESSIVE BLEEDING AFTER TRAUMA

Surgical trauma Dental extraction, tonsillectomy Delayed wound healing Postpartum hemorrhage

JOINT AND MUSCLE BLEEDS

Hemarthroses (bleeding into joints) and spontaneous muscle hematomas
Characteristic of severe plasma protein deficiencies

Characteristic of Hemophilias
Rarely occur in other bleeding disorders
Except severe von Willebrand disease

TYPES OF BLEEDING DISORDERS

Disorder of
Primary platelet plug formation Fibrin formation Premature clot dissolution - fibrinolysis

Spontaneous skin petechiae

Usually severe thrombocytopenia

Spontaneous hemarthrosis
Usually coagulation factor deficiency

TYPICAL SCREENING TESTS FOR BLEEDING DISORDERS

Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT) Quantitative platelet count (+/-) Bleeding Time Test (BTT) (+/-) Thrombin Time

LAB TESTS IN DISORDERS OF PRIMARY HEMOSTASIS

Platlet count Vascular disorder Thrombocytopenia Platlet Dysfunction Normal PT Normal APTT Normal Normal Normal Bleeding time Normal or abnormal Abnormal Normal or Abnormal

Decreased Normal Usually Normal Normal

DISORDERS OF PRIMARY HEMOSTASIS

Disorders of platlets and blood vessels

VASCULAR DISORDERS
Most vascular diseases
Are not associated with platelet or plasma defects Most common symptom
Abnormal bleeding into or under the skin due to increased permeability to blood

Laboratory tests are used to exclude

Coagulation or platelet disorders

Majority of patients
Hemostatic testing is entirely normal, despite a history or physical examination that suggests substantial bleeding

PLATELET DISORDERS
Quantitative
Thrombocytopenia Thrombocytosis

Qualitative Morphologic abnormalities

Macrothrombocytes Microthrombocytes Hypogranular or agranular platelets

THROMBOCYTOPENIA
Platelet count
<150 x 109/L Usually no risk of bleeding unless <50 x 109/L Risk of severe and spontaneous bleeding when platelet count is <10 x 109/L Petechiae Bleeding from mucous membranes

GI, GU tract, etc Bleeding into CNS BT is related to the platelet count unless there is also a concurrent platelet dysfunction

THROMBOCYTOPENIA
Thrombocytopenia may result from
Abnormal platlet distribution Deficient platlet production Increased platlet destruction

PLATELET SEQUESTRATION (DISTRIBUTION DEFECT)

Normally ~30% of platelets held in spleen Splenomegaly/hypersplenism
Up to 90% sequestered May occur in a wide variety of diseases
Infection Inflammation Hematologic diseases Neoplasias

DECREASED PRODUCTION
Failure of BM to deliver adequate platelets to the peripheral blood
Hypoplasia of megakaryocytes
Drug or radiation therapy for malignant disease Acquired aplastic anemia Replacement of normal marrow
Leukemias and lymphomas MDS Other neoplastic diseases Fibrosis or granulomatous inflammation

Ineffective thrombopoiesis
Megaloblastic anemia

DECREASED PRODUCTION
Hereditary thrombocytopenias
Congenital aplastic anemia Wiskott-Aldrich Syndrome (WAS) X-Linked Thrombocytopenia (XLT) Bernard-Soulier syndrome (BSS) May-Hegglin anomaly (MHA) Congenital amegakaryocytic thrombocytopenia (CAMT) Congenital thrombocytopenia with radioulnar synostosis (CTRUS) Thrombocytopenia with absent radii Syndrome (TAR)

INCREASED DESTRUCTION
Immune destruction
Platelets are destroyed by antibodies
Platelets with bound antibody are removed by mononuclear phagocytes in the spleen Anti-platlet antibody tests to identify antibodies on platelets are available

INCREASED DESTRUCTION

 Caused by an autoreactive antibody to the patients platlets

IDIOPATHIC THROMBOCYTOPENIC PURPURA (ITP)

Young children acute and usually transient for 1-2 weeks with spontaneous remission Adults chronic and occurs more often in women Treatment
Corticosteroids Splenectomy Rituximab

ALLOIMMUNE THROMBOCYTOPENIAS
Isoimmune neonatal thrombocytopenia
Maternal antibodies produced against paternal antigens on fetal platelets Similar to erythroblastosis fetalis HPA-1a Most serious risk: bleeding into CNS

ALLOIMMUNE THROMBOCYTOPENIAS
Posttransfusion purpura
More common in females
Previously sensitized, pregnancy or transfusion

Thrombocytopenia
Usually occurs 1 week after transfusion

Transfused and recipients and antigen-negative platelets are destroyed

DRUG-INDUCED THROMBOCYTOPENIAS
Many drugs implicated Same mechanisms as described for drug induced destruction of RBCs Symptoms of excess bleeding
Usually appear suddenly and can be severe

Removal of drug
Usually halts thrombocytopenia and bleeding symptoms

HEPARIN AND THROMBOCYTOPENIA

Heparin associated thrombocytopenia (HAT)
Non-immune mediated mechanism
Develops early in treatment and is benign Heparin causes direct platelet activation
Thrombocytopenia

Immune mediated destruction of platelets

Antibody develops against a platlet factor 4heparin complex
Attaches to platelet surface platelet clearance

MISCELLANEOUS IMMUNE THROMBOCYTOPENIA

Secondary feature in many diseases
Collagen diseases Other autoimmune disorders (SLE, RA) Lymphoproliferative disorders (HD, CLL) Infections
EBV, HIV, CMV, bacterial septicemia

NON-IMMUNE MECHANISMS OF DESTRUCTION

Disseminated intravascular coagulation (DIC) Thrombotic thrombocytopenic purpura (TTP) Hemolytic Uremic Syndrome (HUS) PNH Mechanical destruction artificial heart valves

THROMBOCYTOSIS
platelet count above reference range
Peripheral blood smear
> 20 platelets per 100 x oil immersion field

Result of production by BM (not prolonged lifespan) BM megakaryocytes Primary

Occurs in chronic myeloproliferative disorders and myelodysplasia

Secondary thrombocytosis
Reactive thrombocytosis platelets caused by another disease or condition

Transient thrombocytosis

QUALITATIVE PLATELET DISORDERS

Clinical symptoms vary
Asymptomatic mild, easy bruisability severe, life-threatening hemorrhaging

Type of bleeding
Petechiae Easy & spontaneous bruising Bleeding from mucous membranes Prolonged bleeding from trauma

INHERITED QUALITATIVE PLATELET DISORDERS

Defects in platelet-vessel wall interaction
Disorders of adhesion
von Willebrand disease Bernard-Soulier syndrome
GP-IcIIa; GPVI
Deficiency or defect in plasma VWF Deficiency or defect in GPIb/IX/V

Defects in collagen receptors

Defects in platelet-platelet interaction

Disorders of aggregation
Congenital afibrinogenemia - Deficiency of plasma fibrinogen Glanzmann thrombasthenia
Deficiency or defect in GPIIb/IIIa

INHERITED QUALITATIVE PLATELET DISORDERS

Defects of platelet secretion and signal transduction
Diverse group of disorders with impaired secretion of granule contents Results in abnormal aggregation during platelet activation

Abnormalities of platelet granules

Storage pool deficiency
SPD (grey platlet syndrome) SPD SPD

Defects in platelet coagulant activity

Decreased Va-Xa binding and VIIIa-IXa binding slows normal coagulant response

INHERITED DISORDERS OF PLATLET FUNCTION

VON WILLEBRAND DISESE

ACQUIRED QUALITATIVE PLATELET DISORDERS

Chronic renal failure
Platelet defects associated with uremic plasma

Cardiopulmonary bypass surgery

Thrombocytopenia Abnormal platelet function Platelet defect likely due to
Effects of platelet activation Fragmentation in extracorporeal circulation

Liver disease
Thrombocytopenia due to splenomegaly from portal hypertension

Paraproteinemias
Clinical bleeding and platelet dysfunction are often seen

 Chronic Myeloproliferative Disorders

Can see either bleeding or thrombosis Abnormal platelet function

HEMATOLOGIC DISORDERS THAT AFFECT PLATELET FUNCTION

Leukemias & Myelodysplastic Syndromes

Bleeding usually due to thrombocytopenia Abnormal platelet function

Dysproteinemias
MM and Waldenstroms macroglobulinemia Thrombocytopenia most likely cause of bleeding

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 is the only drug with a definitely established risk of excessive bleeding

DRUGS THAT ALTER PLATELET FUNCTION

Aspirin
Inhibits platelet aggregation Inhibits platelet secretion

Disoders of secondary hemostasis

Disorders of plasma clotting factors

Disorders of Secondary Hemostasis

Disorders of the proteins of fibrin formation (coagulation factors) Disorders of proteins associated with fibrinolysis (increased clot lysis)

Disorders of Secondary Hemostasis

Symptoms of secondary hemostatic disorders
Delayed bleeding Deep muscular bleeding Spontaneous joint bleeding Symptoms common to primary and secondary hemostatic disorders
Ecchymoses GI bleeding Hematuria Hypermenorrhea Gingival bleeding Increased bleeding after tooth extraction Intracranial bleeding Epistaxis

Disorders of Proteins of Fibrin Formation

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

Coagulation screening tests in congenital deficiencies

Platelet count
N

PT

APTT

TT

Congenital Deficiency

XIII, mild deficiency of any factor, plasminogen activator inhibitor-1, 2 anti-plasmin VII

XII, XI, IX, VIII, prekallikrein, high molecular weight kininogen X, V, II

Fibrinogen

A or N

Von Willebrands

von Willebrand Disease

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 cause bleeding by impairing either platelet adhesion or blood clotting

VWD
Three major categories of VWD Type 1 VWD partial quantitative deficiency of VWF Type 2 VWD qualitative deficiency of VWF Divided into 4 variants Type 2A platelet-dependent function Absence of high-molecular weight VWF multimers Type 2B affinity for platelet GPIb Type 2M platelet-dependent function Not caused by the absence of HMW multimers Type 2N Markedly affinity for F-VIII

VWD
Type 3 VWD total deficiency of VWF Types 1 and 2 autosomal dominant inheritance Type 3 autosomal recessive inheritance
Diagnosis Specific tests Quantify VWF and F-VIII activity

VWD Clinical Manifestations

Hemorrhagic tendency is highly variable
Depends on the type and severity of disease Patients with Type 1 and 2 disease
May have mild bleeding symptoms Characterized by hemorrhage from delicate mucocutaneous tissues Epistaxis, easy bruising, GI bleeding, menorrhagia Hematoma and hemarthroses, characteristic of hemophilia A, are not prominent

VWD Clinical Manifestations

Patients with severe type 3 VWD
Severe hemorrhagic tendency Spontaneous hemorrhage Mucous membranes, GI tract Can be frequent and may be life threatening Low F-VIII level Deep hematomas Joint hemorrhages similar to hemophilia

VWD Therapy
Goal
Correct both bleeding time and coagulation abnormalities
Raise both F-VIII and VWF to normal levels
Desmopressin acetate (DDAVP) stimulates release of vWF from endothelial cells Intermediate purity factor VIII which contains intact vWF

Hemophilias
Hemophilia A
Factor VIII Deficiency
Antihemophilic Factor X-linked recessive disorder Most common type of hemophilia

Hemophilias
Hemophilia B
Factor IX Deficiency
Christmas Factor (from family of first patients diagnosed with the disorder) X-linked recessive disorder

Hemophilias
Hemophilia C
Factor XI Deficiency Autosomal recessive disorder seen primarily in the Ashkenazi Jewish population Symptoms range from mild to severe

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

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 Clinical Presentation

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 Treatment
Replacement of clotting factor to achieve hemostasis Various products available

Replacement products benefits vs risks

Blood-born pathogens

Plasma-derived low, intermediate and high purity products Plasma-derived ultrapure products Ultrapure recombinant products
Hepatitis A, B, C, G; HIV, Parvovirus B-19 Thrombotic complications with some F-IX concentrations Development of alloantibody inhibitors

Acquired Disorders
More common than hereditary disorders
Usually involve defects of multiple hemostatic factors Bleeding often simultaneously from >1 site May occur in response to another disease process Can be produced by a variety of mechanisms

Disseminated Intravascular Coagulation

Normal balance of hemostasis is altered Results in the uncontrolled inappropriate formation and lysis of fibrin within the blood vessels Activation of coagulation occurs systemically rather than locally at site of injury Fibrin is deposited diffusely within capillaries, arterioles and venules Clotting proteins, inhibitors and platelets are consumed faster than they are synthesized Acquired deficiency of multiple hemostatic components Fibrinolysis follows fibrin formation Patient generally bleeds spontaneously at the same time that disseminated clotting is occurring

Clinical conditions associated with DIC

DIC - Pathophysiology
Fibrin strands within small vessels result in
Traumatic destruction of RBC
Microangiopathic hemolytic anemia Formation of schistocytes

Fibrin deposition in and obstruction of microvasculature

Tissue anoxia/microinfarcts Renal failure, liver failure, respiratory failure, shock and death

DIC Laboratory Diagnosis

Laboratory diagnosis is difficult
Available tests are nonspecific No single test can establish the definitive diagnosis of DIC PT, APTT, TT prolonged Fibrin degradation products are (+) Platelet count ; platelet function tests abnormal Schistocytes, thrombocytopenia on peripheral blood smear

DIC Therapy
Eliminate underlying cause, if possible
Acute DIC is often self-limited
Will disappear when fibrin is lysed

Replacement therapy
Platelets, RBC, Cryoprecipitate or fresh frozen plasma

Vitamin K Deficiency
Precursor proteins synthesized by hepatocytes Not -carboxylated Ca++-binding sites are nonfunctional Induced functional deficiencies of all vitamin-K dependent proteins Causes of vitamin K deficiency in adults Malabsorptive syndromes Biliary tract obstruction Prolonged broad-spectrum antibiotics Most often seen in newborns Hemorrhagic disease of the newborn Due to newborn hepatic immaturity

Acquired Pathologic Inhibitors

Circulating anticoagulants Usually IgG or IgM immunoglobulins Inhibitors of single factors Patients with inherited factor deficiencies After treatment with replacement concentrates Associated with other conditions Diseases, drugs Occasionally seen in otherwise healthy individuals Interfere with or neutralize clotting factor activity Prolonged screening test not corrected by 1:1 mixture with normal plasma