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c h a p t e r 36

Biochemistry of Hemostasis

Heparin Structure (Repeating Disaccharides)

Pentasaccharide

Sequence (

High Affinity for Antithrombin

)

F I G U R E 3 6 - 1 7

(Also see color figure.) Structure of heparin. Heparin is a polymer of repeating disaccharide units that contain one uronic

acid and one hexosamine residue. The uronic acid residues may be either glucuronic acid or iduronic acid, both of which

are monosaccharide acids that differ in their stereochemistry. The hexosamine residue is glucosamine. Both the uronic

and hexosamine residues can be modified by O- and N-sulfation and the glucosamine residue by N-acetylation. All

heparins bind to antithrombin; however, heparin molecules that contain a unique pentasaccharide sequence bind with

particularly high affinity (high-affinity heparins). Approximately 30% of the heparin molecules present in the commonly

used therapeutic heparins have high affinity for antithrombin.

Activation of plasminogen occurs as a result of the bind-

ing of both plasminogen and t-PA to fibrin, which results

in a nearly 400-fold decrease in the

Km

for the activation

reaction, from about 65

pM

to 0.16 /zM. In addition to this

enhancement of ES binding, the common binding to the

same “surface” localizes the activation reaction to fibrin.

Binding to fibrin occurs via the lysyl residues in fibrin and,

as a result of fibrinolysis, plasmin proteolysis of Lys-X

peptide bonds increases the number of available sites to

which the plasminogen and t-PA can bind. Kringles 1

and 4 of plasminogen and plasma bind to fibrin via their

lysine binding sites. The activation of plasminogen by

t-PA and u-PA is opposed by two protein inhibitors, plas-

minogen activator inhibitors 1

and 2 (PAI-1 and PAI-2;

Figure 36-18). PAI-1

inactivates t-PA very rapidly, but less

so when the t-PA is bound to fibrin. Higher than normal

concentrations of PAI-1 are associated with the occurrence

of thrombosis, indicative of the necessity for balance be-

tween tPA activation of plasminogen and t-PA inactivation

by PAI-1.

Plasminogen can be activated rapidly by the formation

of a “stoichiometric” complex between the plasminogen

molecule and proteins from several strains of hemolytic

Streptococcus

and

Staphylococcus.

The “streptokinase-

plasmin” complex (SK-plasmin) converts plasminogen to

plasmin as well as digesting fibrin. In contrast to normal

plasmin, SK-plasmin is not inhibited by a^-antiplasmin.

Streptokinase-plasmin(ogen) is used therapeutically in

fibrinolytic therapy to remove thrombi. Another therapeu-

tically useful product has been prepared from SK-plasmin.

In this product, the active Ser of the plasmin is reacted with

an acylating chemical to form an unstable acyl ester. Slow,

spontaneous hydrolysis of the acylated SK-plasmin results

in a more sustained level of SK-plasmin in the circulation

during fibrinolytic therapy.

Plasmin is inactivated by a

2

-antiplasmin in a very rapid

reaction;

kjIKt

is approximately 2 x 10

7

M

- 1

s

- 1

{k^K^

is analogous to

kJKm,

the best indicator of the efficiency

of an enzyme-catalyzed reaction). Blocking of the kringle

Lys binding sites reduces the rate of plasmin inactivation.

This is most evident from the “protection” of plasmin that

occurs when the plasmin is bound to the fibrin strands.

This protection tips the balance between the proteolytic

action of plasmin on its fibrin substrate and its inactiva-

tion by o'

2

-antiplasmin to proteolysis. The half-life (time

for half of the plasmin to be inactivated by a

2

-antiplasmin)

is greater than

1 0

s for plasmin bound to fibrin, while it is

approximately 0.1 s in the absence of fibrin. This is another