Biochemistry of clotting Flashcards
Plasma
Blood + anti-coagulant, then spun down
Clotting factors still in zymogen form
Serum
Blood is allowed to clot, then spun down
Clotting factors inactivated, fibrinogen depleted
Platelets
formed from megakaryocytes (WBCs synthesized in marrow) non-nucleated 2-3 microm in diameter half life: 5-9 days 150-400 x 10^9/L activated by thrombin or ADP
Physiological events in thrombosis
Endothelial damage
platelet adhesion
platelet aggregation
Fibrin clot formation
Overview of clotting cascade
Initiator (tissue damage)
activation of clotting factors
Thrombin catalyzes fibrinogen (soluble) –> fibrin (insoluble)
Extrinsic pathway
Initiation
components are platelet-membrane bound
Vascular injury Tissue factor Factor VII- tissue factor --> Factor VIIa-tissue factor VIIa catalyzes Factor IX --> Factor IXa VIIa catalyzes Factor X --> Factor Xa
Common pathway
Factor Xa catalyzes prothrombin –> thrombin
Thrombin catalyzes V –> Va, which catalyzes above
Thrombin catalzes fibrinogen –> fibrin
Fibrin + Factor XIIIa (converted by thrombin) –> cross-linked fibrin
Intrinsic pathway
Propagation
Factor XI –> XIa
XIa catalyzes IX –> IXa
IXa catalyzes X –> Xa
Thrombin catalyzes XI –> Xia, VIII –> VIIIa
Fibrinogen
6 polypeptides : a2b2gamma2
shaped like a dumbell
Three globular domains linked by triple helixes
Fibrin formation
Fibrinogen is cleaved at 4 sites (N-termini)
Fibrinopeptides A&B removed, leaving behind alpha and beta knobs
Fibrin polymerization
New N-termini on activated fibrin can bind terminal domains on other fibrins
Dimer still soluble
polymer insoluble
Fibrin cross-linking
Introduced by XIIIa
Covalent bonds between adjacent terminal domains
XIIIa also crosslinks fibrin clot to surrounding tissue
Role of vitamin K in clotting
Vitamin-K dependent carboxylation required for biological activation
Normally, gut flora synthesis sufficient
Vitamin K injection in newborns
Glu –> Gla
Gla protein - role in clotting
Gla binds Ca, conformation change
Ca-Gla-proteins can bind phospholid membranes (supplied by activated platelets)
Ensures that fibrin formation occurs at the site of injury instead of flowing in blood
Gla required for membrane binding (need vit K) of VII, X, IX, prothrombin ( = vit K proteins)
allow concentration, orientation, localization
Factor XII
Deficient patients don’t bleed
Probably has non-coagulant functions
Minor role in hemostasis compared to TF-fVIIa-TFPI complex
Activated by poly-phosphate on the activated platelet surface
Resulting XIIa activates XI –> XIa, generating thrombin for further platelet activation after TF-fVIIa-TFPI complex is formed
XII deficiency protects against arterial thrombus formation
Termination of clotting
dilution
trapping of proteases in fibrin clot
serine protease inhibitors (Serpins) such as antithrombin bind to active sites of thrombin and forms inactive complex
Antithrombin deficiency: risk of thrombosis
Heparin accelerates antithrombin-thrombin reaction (anticoagulant)
Thrombomodulin - Protein C - Protein S pathway
Thrombomodulin appears on endothelial cell membrane proteins
converts thrombin from a procoagulant to an anticoagulant
which then activates Protein C (protease) with protein S
leads to inactivation of Va, VIIIa
Plasminogen activation
Activator (protease - tPA, uPA, streptokinase/staphylokinase)
converts plasminogen –> plasmin (enzyme)
Role of plasmin
cleaves triple helix of the fibrin polymer
forms D-dimers, which are soluble and cleared by the liver
Lab coagulation tests
Prothrombin time - INR
Activated partial thromboplastin time (APTT) - also PTT
Prothrombin time
Plasma incubated with thromboplastin (phospholipid + tissue factor), then Ca added
Measure clotting time of extrinsic & common pathway
10-13 s but varies
INR
Prothrombin time (in seconds), varies among labs Differences in batches of tissue factor
INR = [patient time/reference time ]^ISI ISI = international sensitivity index
Normal people, INR = 1
anticoagulant drugs >1
Activated partial thromboplastin time
Plasma + kaolin + partial thromboplastin (phospholipid, no tissue factor) , add Ca
Measure clotting time (intrinsic/common pathways)
24-37s, varies
