Hemostasis & Thrombosis Beth A. Bouchard BIOC 212: Biochemistry of Human Disease Spring 2006

Hemostasis & Thrombosis

Beth A. BouchardBIOC 212: Biochemistry of Human Disease

Spring 2006

HEMOSTASIS

Hemorrhage Thrombosis

Hemostasis

HEMOSTASIS (CONT.)

1). INITIATIONVessel wall – endothelial cells and subendothelial

components

2). LOCALIZATIONPlatelets – circulating cellular elements

3). PROPAGATION/AMPLIFICATIONPlasma coagulation proteins (factors)

4). TERMINATIONPlasma coagulation protein inhibitors

5). ELIMINATIONFibrinolytic system

Vessel Wall: Endothelial cells

Antithrombotic Attributes of Vascular Endothelium

Vessel Wall (cont.)

Response to Vessel Wall Injury: Platelet adhesion

• Exposure of flowing blood and platelets to subendothelial components

• Platelets bind to the subendothelial collagen bound to von Willebrand factor (vWF), which is secreted from endothelial cells directly into the subendothelial space or adsorbed from plasma following endothelial cell secretion

• vWf also binds directly to platelets via glycoprotein Ib-IX

PLATELETS

Platelets adhered to damaged endothelium

Platelet Plug Formation = 1° hemostasis

Platelet Plug Formation: Platelet activation

• Activated via their interaction with subendothelial collagen

• Additional platelet agonists include ADP, epinephrine, thrombin, immune complexes, and high shear stress – all of the compounds interact with specific platelet membrane receptors

• Several platelet activation pathways are initiated

Platelet Plug Formation: Platelet activation events

• Platelet shape change: extend pseudopodia, which facilitates aggregation and coagulant activity

• Release of alpha and dense granule contents including a number of compounds involved in hemostasis (eg ADP, factor V and fibrinogen)

• Aggregation

Response to Vessel Wall Injury: Vasoconstriction

• Temporarily reduces local blood flow and hence, blood loss

• Mediated in part by serotonin and thromboxane A2 (TXA2) from activated plateletsSerotonin is released from platelet dense granules

TXA2 is a product of platelet prostaglandin metabolism

Activated platelets

Platelet Plug Formation: Platelet aggregation

• Platelet activation results in the functional expression of membrane receptors normally expressed in a non-functional state (glycoprotein IIb-IIIa)

• Fibrinogen from the plasma or released from activated platelet alpha-granules binds to activated glycoprotein IIb-IIIa membrane receptors effectively bridging platelets to each other

Platelets adhered to and aggregated upon collagen

Platelet Plug Formation


components





HEMOSTASIS

BLOOD COAGULATION

BLOOD COAGULATION (CONT.)

• Deficiencies in all of the factors, except factor XII, lead to a bleeding tendency in the affected individual

• Described as a ‘waterfall’ or ‘cascade’ sequence of zymogen (pro-enzyme) to enzyme conversions, with each enzyme activating the next zymogen in the seqeunce

• Activated factor enzymes are designated with an “a”, e.g. factor Xa

Common constituents of coagulation complexes

Vitamin K-dependent (VKD) zymogen

Ca2+

Appropriate membrane surface

- activated platelets (VIIIa/IXa complex, Va/Xa complex)

- subendothelial cells, typically fibroblasts (TF/VIIa complex)

Protein cofactor



Ca2+




Protein cofactor

Functional Domains of the Vitamin K-dependent Zymogens

Gamma ()-carboxyglutamic acid

Formation of Gla residues subsequent to protein synthesis (post-translational)

• Group of related, fat soluble compounds, which differ in the number of side-chain isoprenoid units

• Plant derived (vitamin K1) and synthesized by intestinal bacteria (vitamin K2)

H2



Protein cofactor

Ca2+




Ca2+

FXaCa2+ 30

Ca2+

FXa

FVa HC

FVa LC

Ca2+

300,000

-Thrombin

ProthrombinaseComponents

Relative Rateof Prothrombin

Activation

FXa 1Prothrombin

Ca2+

FXa

Prothrombinase

FVa HC

FVa LC

Ca2+

FXa 300FVa HC

FVa LC

Ca2+

Relevance of complex formation and its constituents



Protein cofactor

Ca2+




** Express anionic phospholipids and membrane receptors for coagulation proteins.

In platelets, the expression of this membrane surface is activation-dependent.

Activate platelets

HEMOSTASIS (CONT.)


components





INHIBITORS

INHIBITORS (cont.)

FIBRINOLYSIS

FIBRINOLYSIS (CONT.)

Platelet Plug Formation

• Measured clinically as the bleeding time

• Normal bleeding time is from 2 – 10 min

• Usually the bleeding time is sufficient to detect defects of platelet adhesion and aggregation, in which it is prolonged

Intrinsic Pathway of Blood Coagulation

• No factors extrinsic to the blood are involved

• Clinical test to assess the functionality of this pathway is the activated partial thromboplastin time (aPTT)– Kaolin and cephalin are added to the test plasma sample– The normal range is ~30 – 50 seconds (varies slightly

depending on the laboratory)– Prolongations in the aPTT are observed in deficiencies of

factors XI, IX, VIII, X, and V, prothrombin, or fibrinogen.– Used to test for common congenital hemophilias

(deficiencies in IX, VIII, or XI) and to monitor heparin treatment

Extrinsic Pathway of Blood Coagulation

• Extrinsic refers to tissue factor, which is expressed on subendothelial cells

• Clinical test to assess the functionality of this pathway is the prothrombin time (PT)– Lipidated tissue factor is added to test plasma sample– The normal range is ~10-15 seconds (varies slightly

depending on the laboratory)– Prolongations in the PT are observed in deficiencies of

factors VII, X, V, prothrombin, or fibrinogen.– Used to test for the rare congenital deficiencies in these

factors: More often it is used to diagnose acquired bleeding disorders resulting from vitamin K deficiency, oral anticoagulants (e.g. warfarin), and liver disease

Thrombin Time (TT)

In this test, thrombin is added to plasma

– The normal range is ~10-15 seconds (varies slightly depending on the laboratory)

– Prolongations in the TT are observed in congenital fibrinogen deficiency or acquired fibrinogen deficiency resulting from consumption of fibrinogen in DIC (disseminated intravascular coagulation), or may occur following treatment with fibrinolytic drugs

Hemorrhage

Bleeding disorders can span the spectrum from weeping blood vessels to full-fledged internal and external hemorrhage

Genetic defects:platelet abnormalitiesblood vessel wall abnormalitiesclotting factor deficiencies (hemophilias)excess clot breakdown (fibrinolysis)

Acquired defects:liver disease (site of clotting factor synthesis)vitamin K deficiencyautoimmune disease (platelet destruction)trauma

Disorders of Platelet Adhesion or Aggregation

• Affecting constituents of the vessel wall

• Affecting the ability of the platelet to interact with the subendothelium at sites of blood vessel injury

• Affecting the ability of the platelet to interact with other platelets

Vessel Wall Defects

• von Willebrand’s disease: a group of autosomal dominant disorders that result in reduced or abnormal synthesis of vWF

• Defects in collagen synthesisEhlers-Danlos Syndrome: congenital defect in collagen synthesis

Scurvy: results from vitamin C (ascorbic acid) deficiency, which is involved in collagen synthesis

Excess exogenous or endogenous corticosteroids: also leads to acquired deficiency in collagen synthesis

Platelet Defects

• Bernard-Soulier Syndrome: expression of low levels of or defective glycoprotein Ib-IX on the platelet surface

• Glanzmann’s thrombasthenia: expression of low levels of or defective glycoprotein IIb-IIIa on the platelet surface

Other Defects

• Fibrinogen: deficiency of or production of abnormal protein

• Acquired disorders include low platelet count (thrombocytopenia) as a result of defective formation of platelets by the bone marrow or excessive destruction of platelets

Hemorrhage


Genetic defects:platelet abnormalitiesblood vessel wall abnormalitiesclotting factor deficiencies (hemophilias)excess clot breakdown (fibrinolysis)

Acquired defects:liver disease (site of clotting factor synthesis)vitamin K deficiencyautoimmune disease (platelet destruction)trauma

Vitamin K deficiency

• Deficiency of vitamin K is rare because of its wide distribution in nature, and its production by intestinal bacteria

• Found in individuals with liver disease and fat malabsorption - it is associated with bleeding disorders

• Newborn infants (especially preemies) are also at risk- Placenta is insufficient in the transfer of maternal vitamin K- Concentration of circulating vitamin K drops immediately after birth, and it recovers upon absorption of food- Gut of the newborn is sterile

Thus, newborns are given an injection of vitamin K following birth.

Hemophilias A and B

• Hemophilias A and B are cause by deficiencies in factors VIII or IX, respectively

• Affect ~1 in 10,000 males

• Inherited as a recessive X-linked trait (Mom would be an unaffected carrier)

• Treated by administration of factor VIII or factor IX concentrates

• Recombinant factor VIII or XI• Gene therapy trials

Hemorrhage


Treated by factor replacement

Thrombosis

Thrombosis can be manifested as a transient, short-term or episodic event in individuals with chronic or recurring clotting. It is the major cause of both stroke and heart attacks.

Genetic defects:clotting factor INHIBITOR deficienciesdecreased fibrinolysis

Acquired defects:atherosclerosis

Pharmacologic Approaches to Prevent Thrombosis

Antiplatelet agents - block activation, aggregation or intraplatelet agonist synthesis

Effective anticoagulant therapy includes both antiplatelet and antithrombin agents

Antiplatelet Drugs

Blood “thinners” - coumadin (warfarin): inhibition of “gla” formation in the liver

Coumadin blocks reformation of reduced vitamin K, which essentially stops the post-translational modification of the glutamic acid residues at the amino-termini of the VKDP’s, since vitamin K is oxidized during the reaction.

Blood “thinners” - heparin: potent cofactor for ATIII-catalyzedinhibition of procoagulant serine proteases

Snakes

Leeches

Blood-sucking Insects

Documents

Hemostasis & Thrombosis Beth A. Bouchard BIOC 212: Biochemistry of Human Disease Spring 2006