Normal Coagulation

Coagulation

delicate balance between prevention of blood loss and maintenance of fluidity

complex interaction between platelets, clotting factors present in plasma, tissue factors

most interaction is at platelet surface

Fluid Phase "Coagulation Cascade"

series of proenzymes (zymogens) activated to enzymes (active form lower case "a") – most are serine proteases

all enzymes are made in liver except factor VIII (possibly made by mononuclear phagocytic cells or sinusoidal endothelial cells)

several (prothrombin, factors VII, IX and X) require vitamin K (from food and bacteria in gut) for synthesis

add second carboxylic acid to gamma carbon of C-terminal glutamic acid to form carboxyglutamic acid

end result: prothrombin Þ thrombin, which cleaves fibrinogen (soluble) Þ fibrin (insoluble) Þ clot

Prothrombin – produced in the liver – one of the vitamin K dependent coagulation factors

activation mediated by factor Xa, which splits 2 peptide bonds to release thrombin
reaction enhanced 300,000 fold by prothrombinase complex on phospholipid surface (platelet) with factor Va and Ca⁺⁺

Ca⁺⁺ binds C-terminal carboxy group of carboxyglutamic acid

Thrombin – hydrolytic enzyme (split arg-gly of fibrinogen)

hydrolyzes other important coagulation factors leading to their activation (factors V, VIII, and XIII), stimulates platelets

Fibrinogen (340 kD) – made in liver, 150-400 mg/dl in plasma

three pairs of polypeptide chains (A, B, D) held together by disulfide bonds
during activation, N-terminal end of A and B chains cleaved off to produce fibrinopeptide A and B, respectively
release of the fibrinopeptides produces fibrin monomers (form loose dimers with unaltered fibrinogen)

as more fibrin monomers are generated, dimers dissociate, monomers spontaneously polymerize
factor XIII and calcium stabilize fibrin by cross-linking fibrin polymers by forming covalent bonds

Mechanisms of Clotting

(1) Intrinsic System

Contact Phase of Activation–blood contacts negatively charged surface (collagen or subendothelial basement membrane)

collagen or basement membrane causes conformational change in factor XII, activating it

four proteins involved: Factor XII (Hageman factor), factor XI, prekallikrein and high mol. wt. kininogen

factor XII cleves then cleaves factor XI to form XIa, which is also adsorbed on the surface

this is accellerated by kallikrein (made from prekallikrein by factor XII)

kallikrein also induces chemotaxis of leukocytes and participates in activation of factor XI

high mol. wt. kininogen (cofactor, activated by kallikrein) may also be necessary for XI Þ Xia

produces kinins (contract smooth muscle, dilate small blood vessels, incr. vascular permeability, pain)

factor XIa cleaves factor IX to IXa by Ca⁺⁺-dependent process

factor IXa, factor VIIIa (made from factor VIII by thrombin), and Ca⁺⁺ interact on phospholipid (platelet membrane)

form a complex that activates factor X to factor Xa

factor IXa is the active enzyme; rest facilitate catalytically efficient complex

factor Xa interacts with factor Va, phospholipid and Ca++ to release thrombin from prothrombin

phospholipid binds carboxy glutamic acid residues of factor VII and X by linking through Ca⁺⁺ ions

(2) Extrinsic Pathway (tissue factor pathway)

occurs when blood is exposed to tissue extracts that contains tissue factor (a transmembrane protein)
in the presence Ca⁺⁺, tissue factor reacts with factor VII to form VIIa (stoichiometric)

complex of tissue factor/phospholipid/factor VIIa/ Ca2+ activates factor X to generate factor Xa

factor Xa goes on to release thrombin as before

this tissue factor pathway is now thought to be the principal initiating pathway of blood coagulation

Exceptions to the Pathways

factor VIIa and tissue factor can also convert IX to IXa, producing thrombin by the intrinsic method

as soon as small amount of thrombin is generated, tissue factor pathway inhibitor (TFPI) shuts off this pathway

Regulating Thrombin Generation – Natural Anticoagulants

antithrombin – neutralizes thrombin and several other serine proteases (esp. Xa, also IXa, XIa, XIIa, kallikrein)

binds thrombin and Xa in the presence of Heparan sulfate and heparin to prevent activity therefore controlling conversion of fibrinogen to fibrin
interacts with other glycosaminoglycans (dermatan sulfate, chondroitin sulfate)

induces expression of prostacyclin (PGI₂ ) on endothelium, which in turn inhibits platelet aggregation

Protein C, Activated Protein C (APC), and Protein S – vitamin K-dependent

thrombin not used to make fibrin binds thrombomodulin (TM) on endothelial surface – protein C Þ APC

Protein S – cofactor for the generation of APC

APC inactivates FVa and FVIIIa

tissue factor pathway inhibitor (TFPI) – associated with plasma lipoproteins

inhibits tissue factor/factor VIIa complex to inhibit activation of factor X and factor IX

C1 inhibitor – inhibits factor XIIa, factor XIa, plasmin and kallikrein

additionally, the liver removes active enzymes at a constant rate

Fibrinolytic System – Dissolving Clots to Prevent Ischemia

regulates plasmin (digests clots) formed from its precursor, plasminogen

Plasmin – serine protease comprised of two chains held together by a disulfide bond

cleaves fibrin to soluble fragments
cleaves fibrinogen producing early fragments (X and Y, themselves anticoagulant) and later products (D and E)
cleaves factor VIII, vWF, factor V and other clotting factors
activity physiologically regulated by the plasma inhibitor alpha 2-antiplasmin – binds lysine residue on plasmin
Plasmin can be generated by three distinct pathways:

(1) contact system itself – kallikrein (made by factor XII; see above) can weakly activate plasminogen to plasmin

(2) t-PA (tissue plasminogen activator) – main system – released from vascular endothelium – very high affinity for fibrin

inhibited by plasminogen activator inhibitor-1 (PAI-1), made by endothelium, hepatocytes, fibroblasts, platelets

(3) Urokinase – found in urine (related to bacterial product, streptokinase)

Laboratory Evaluation

to prevent coagulation, simply chelate Ca⁺⁺ by citrate (coagulation assays) or EDTA (blood counts)

plasma can be analyzed by centrifuging anticoagulated blood to produce coagulation factors (in supernatant)

serum can be analyzed by centrifuging non-anticoagulated blood (which lacks fibrinogen and factors V and VIII)

Evaluation of the clotting cascade

(1) Activated Partial Thromboplastin Time (APTT) – measures intrinsic and common pathways of coagulation

clotting time of a mixture of citrated plasma, phospholipid, Ca⁺⁺ and contact activation agent (ellagic acid, kaolin)
prolonged in patients deficient in any plasma clotting factor except factor VII or XIII
may also be prolonged in patients with circulating anticoagulants or inhibitors of coagulation

(2) Prothrombin Time (PT) – evaluates extrinsic and common pathways of coagulation

clotting time of citrated plasma, tissue thromboplastin and Ca⁺⁺ ions
will be prolonged by deficiencies in factor VII, X, V, prothrombin and fibrinogen
may also be prolonged by inhibitors to the extrinsic or common pathway coagulation factors

(3) International Normalized Ratio (INR) – ratio of PT to mean PT (adjusted by ISI)

ISI, international sensitivity index, adjusts for different thrombin used in the test
prothrombin ratio (patient’s PT/mean normal PT)^ISI

(4) Thrombin Time (TT) – clotting time after adding thrombin to citrated (anticoagulated) plasma

prolonged in patients with either quantitative or qualitative abnormalities of fibrinogen
patients on heparin will have prolonged thrombin times.