1-22-10 The Formation of the Hemostatic Plug 1.

Prinicples of normal hemostatic regulation in vivo (hemostasis) and how this differs from the clotting cascade (blood clotting). Blood clotting is a test tube phenomenon! Hemostasis is the in vivo maintenance of vascular integrity and fluidity via site-specific generation of THR !"#$% Ho& is it that thrombin can be generated at localized sites of vascular in'ury( The coagulation factors actually get locali)ed onto receptors on endothelial cells* platelets* and monocytes% !oreover* these factors are in )ymogen form and &on+t be activated until necessary% 2. Understand the physiologic se uence of events that lead to the formation of a stable hemostatic plug after in!ury to a blood vessel. Really* the t&o ,ey players you should remember for hemostatic plug formation are tissue factor and factor "##a% #n greater detail$issue factor is a transmembrane protein found on endothelial cells* fibroblasts* glial cells* and macrophages.monocytes% /0pression on those tissue occurs after perturbation by cyto,ines* endoto0in and gro&th factors 1i%e% during injury2% nce e0pressed* it can bind to activated platelets via P%selectin. This provides the lin, bet&een vessel damage and 1ultimately2 thrombin damage% The main function of TF is to serve as the catalytic cofactor for FVIIa% 3##a is only active &hen bound to TF! Thus* e0cesses of TF can result in lots of clotting* either in the form of &#' or locali(ed thrombosis% For e0ample* infection causes the release of cyto,ines due to e0cess e0pression of tissue factor on endothelial cells and monocytes.macrophages% Typically* you+ll see increased TF levels from trauma or cancer cells% #n addition to TF imbalances* F3## levels can cause problems% 4 F3## deficiency results in e0cessive bleeding after trauma or surgery% 4n e0cess of F3## increases the ris, of !#% The ability of F3## to bind locali)ed sites and become specifically activated is the common underlying theme of hemostatic plug formation% Patients &ith hemophilia are deficient in F3### 1hemophilia 42 and F#5 1hemophilia "2* not TF* &hich &e+ve 'ust established as the initiator of hemostasis% 6hy is it then that hemophilia patients bleed( #n vivo* TF-F3##a comple0 that binds to F5a is rapidly inactivated by TFP# 1Tissue Factor Path&ay #nactivator2% Thus* TF-F3##a instead proceeds through FIX forming the $enase 'omple)% The activated F#5a then binds its cofactor *"###* forming F3###a% The comple0 of F#5a and F3###a binds F5* producing F5a% #n summary7 TF-F3##a 8 F#5->Tenase comple0 8 F3### 8 F5->coagulation 9eficiencies in F#5 or F3### 1hemophilia b and a* respectively2 result in severe hemorrhagic disorders% :eep in mind that F3### is protected by v6F in circulation% 4 deficiency in v6F &ill

result in a deficiency of F3###* &hile an increase in v6F &ill also cause an increase in functional 3###% "ecause v6F is an acute phase reactant* its levels &ill go up &ith inflammatory disorders% F3### &ill also go up &ith it% High levels of F3### result in venous thrombosis and stro,e% v6F levels drop &ith v6illlebrand+s 9isease* resulting in bleeding% The "itamin +%dependent coagulation proteins are ##* 3##* #5* 5* Protein ;* and Protein <% 1=1,-2 'hevy .ilverado>2 3itamin : is a cofactor re?uired for the normal function of glutamyl carbo)ylase* a liver en)yme% @lutamyl carbo0ylase produces gamma carboxyglutamic acid on ##* 3##* #5 and 5% This process recruits calcium* &hich induces the functional conformation in the vitamin :-dependent proteins% The main function of calcium is to change the conformations of the 3it :dependent coagulation proteins into functional beings% ;alcium binding re?uires gamma carbo0yglutamic acid* &hich is produced by glutamyl carbo0ylase% @lutamyl carbo0ylase re?uires vitamin :% 3itamin : 9eficiency is rare and occurs in A &ays7 broad-spectrum antibiotics* 6arfarin.coumadin 1&hich is a 3it : antagonist!!2* fat malabsorption* and hemorrhagic disease of the ne&born% When asked what activates prothrombin->thrombin the answer is !"# only F$a% #t is the prothrombinase comple) consisting of F5a* F3a* and cell receptor 1phospholipids2% 9eficiencies of either F5 or F3 leard to severe hemorrhagic disorder% *actor " /eiden is a common mutation in F3 at amino acid B0C resulting in production of a protein that%s resistant to &'( )activated protein (* degradation+ ;onse?uently* it is an inherited thrombotic disorder% #n contrast to Factor 3 Deiden* there can be disorders of protein ; and protein < itself% Protein ; and Protein < &or, together to proteoly)e F"a and F"###a% 'oumadin necrosis occurs &hen &arfarin.coumadin is given to patients &ith severe protein ; or < deficiencies% #t is characteri)ed by cutaneous thrombosis 1remember &arfarin is a vit : antagonist2% Purpura *ulminans refers to children born &ith a complete deficiency of either Protein ; or Protein <% #t is fatal at birth% $hrombin% #t has three main functions 1mainly acting as a mitogen and an en)yme27 #nitiates hemostatic plug #hrombin is the most potent activator of platelet aggregation% This involves activating platelets so that they e0press @P ##b.###a% #t also proteolytically cleaves fibrinogenfibrin* releasing fibrinopeptides 4 and "% Fibrin monomer polymeri)es via H-bonding% *0###a stabili)es the crosslin,ing in the fibrin clot% #t is an endo-gammaglutamine-lysine transferase that covalently crosslin,s fibrin polymer* fibrin to platelet fibrinogen* and platelet surface proteins into a stable clot% Thrombin activates F5###F5###a%

Functional or ?uantitative deficiency in 5### leads to bleeding disorder characteri)ed by lesions that stop bleeding and then rebleed 1slo&-healing &ounds2% 6omen &ill have difficulty maintaining pregnancy% Euanititative deficiency in fibrinogen is associated &ith bleeding disorder &hen patient has surgery or trauma Eualitative deficiency in fibrinogen is associated &ith bleeding or thrombotic disorder% 1ntithrombin ### inhibits the en)ymatic activity of thrombin% #t is only active &hen associated &ith heparin-like ,&,s* &hich are located on the endothelium and subendothelium 1thus* biologically active antithrombin is present at the site of thrombin generation2 9eficiency in antithrombin causes an inherited thrombotic disorder 9isorders of thrombin & lack of prothrombin is incompatible with life Dastly* thrombin also directly activates F"### and F"* thereby amplifying its o&n generation% #nitiates tissue repair Thrombin stimulates release of P&2* and P&32* 1platelet derived endothelial gro&th factor2 from platelets% #t also induces monocyte4macrophage chemota)is and mitosis of smooth muscle cells% #nitiates mechanisms that maintain vascular fluidity and limit hemostatic plug e0tension% Thrombin induces endothelial cells to release protein5 prostaglandins4cyclin 1inhibit platelet aggregation25 endothelial rela)ing factor and nitric o)ide 1inhibit platelet aggregation and dilate blood vessels2% Thrombin also induces uP1 and tP1 release from endothelial cells% They generate plasminogenplasmin* &hich digests fibrin% #n addition* thrombin induces uP4 receptor e0pression on endothelial cells* monos* and macrophages to increase plasmin generation% Thrombin bound to thrombomodulin activates 'rotein (% 1P' inactivates F3###a and F3a and enhances firbinolysis by inactivating '&I--% 4nd finally* thrombin mediates platelet mediated clot retraction+

4 fe& additional notes regarding the intrinsic path&ay <evere factor 0# deficiency is associated &ith mild-moderate bleeding disorder F5# has an accessory role &hen the hemostatic system is stressed by surgery or trauma% #t probably provides an amplification loop for hemostatic plug formation &hen higher levels of thrombin formation are needed &ith ma'or vascular in'ury% F5a actiaves F#5 &ithout any cofactors or surfaces% The physiological activator of F5# is thrombin 1not F5##a2%