BLOOD COAGULATION: AT

THE INTERFACE BETWEEN

PHYSICAL AND CHEMICAL
KINETICS
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FUSTER et al, 2005
FUSTER et al., 2005
Local homeostasis maintained by continuous adjustments to changes

threshold
Tissue PERTURBATION

RESPONSE
REACTION
RATE CHANGE
“COAGULATION” “INFLAMMATION”
Hemostasis Immunity

thrombophilia hypersensitivity
hemophilia autoimmunity
Disseminated coagulation
Immunodeficiency
 PLASMA COAGULATION/FIBRINOLYSIS

FLOW PROCOAGULANT
SURFACES
 COAGULATION CONTROL
INHIBITORS
HEPARIN (Glycosaminoglycans)
Warfarin (Vit.-K antagonist)
Anti-platelets

PROCOAGULANTS
COAGULATION FACTORS
BLOOD TRANSFUSIONS

STATINS ?
Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A
Reduce cholesterol levels and cardiovascular disease
but
Under the best circumstances 2/3 cardiovascular events remain.
 Contact activator
“autoactivation of fXII
Bradikinin -tPA Pre to kallikrein
H-kininogen to bradikinin
Plasminogen
Fibrin B1 and B2 receptors
Plasmin Of endothelial cells

FIBRINOLYSIS PLA2->PG->cAMP
Hereditary
angioedema Ca2+->eNOS->NO-> cGMP

RELAXATION
Spaan J. A. E. et al., 2003
Spaan J. A. E. et al., 2003
Spaan J. A. E. et al., 2003
Spaan J. A. E. et al., 2003
Spaan J. A. E. et al., 2003
Spaan J. A. E. et al., 2003
A + B ↔ AB →PRODUCT
(1nmol ~ 1014 molecules)

FLUX :
convective → pressure gradient

diffusive→ concentration gradients

FLOW-DEPENDENT TRANSFER?

SURFACE DIFFUSION ON CELLS?

PROTOTYPE REACTION SCHEME

KA Kf
A+B [AB] PRODUCTS

Kd

K eff = KA.Kf / Kd + Kf

K f >> Kd K eff = K A
MODULAR STRUCTURE OF COAGULATION ZYMOGENS

Growth factor-like domain

Gla Domain
Serine proteinase Domain
Aspartic acid
Gamma-carboxylated
Histidine
Glutamic acid residues
SERINE
DIFFUSION-CONTROLLED REACTIONS IN BIOLOGICAL SYSTEMS

Soluble enzyme kinetics

Steady-state diffusion-controlled reaction (Smoluchowski)

Influence of intermolecular forces (Kramers, Debye)

Hydrodynamics effects (Friedman, Deutch)

(osmotic stress techniques?)

Convective flow (Levich, Delichatsios)

Diffusion toward an array of reactive traps. Spherical

Influence of chemical reaction on diffusion in the reactive system

Mean first passage times.

 Glycosaminoglycans role in the local
regulation of interstitial fluid volume

Local hydration = competitive affinity of macromolecules for water

Glycosaminoglycans form hydrophilic gels that store “dehydration

energy”
In vivo manifested as a dehydrating potential.
In vitro detected as swelling pressure

CARTILAGE
VASCULATURE
Polymer rubber elasticity

Polymer­polymer 
affinity
H20

H+   
Pressure
OSMOTIC FORCES
GALACTOSAMINOGLYCANS
 K1/2 Vsat

AT + GAG AT·GAG+fXa AT·GAG-fXa

GAG

AT-fXa
 Antithrombin surface 1.4 Å  under  4.0 Å

 
Water activity  

ΔG/Π = ΔV
 20

18

16
DS (0.5 atm)
DS (standard)
14 DD (0.5 atm)
DD (standard)

12 Heparan (0.5 atm)

Factor Xa nM

Heparan (standard)

10

0
0 5 10 15 20 25 30 35 40 45 50
TIME (seconds)
 20.2

20

19.8

19.6
G ( kcal/mol)

19.4

19.2

19

18.8
0 .5 1 1.5 2 2.5
Osmotic Pressure (atm)
 TABLE 1
(kcal/mol/atm) (water mol/mol)
Probe radius, (Å)
PEG: 300 4 -0.072 ± 0.011 165 ± 25
600 7 -0.221 ± 0.060 508 ± 138
1500 9 -0.336 ± 0.031 772 ± 71
3400 17 -1.16 ± 0.11 2667 ± 252
8000 26 -1.083 ± 0.207 2489 ± 475
Dextran T10 24 -1.287 ± 0.113 2958 ± 260
PVP 40 NA - 1.314 ± 0.176 3028 ± 404
Water Transfer by Osmotic Stress Technique with Polymers of Different Size and Chemical Structure
A PI B C
C-sheet RCL

hH
B-sheet

hG

hD
hA
hB
hl

hE

hF A-sheet
hC
 140
1

120
1

1
100
RATE s-1 X103

80

60

40

20

0
0 20 40 60 80 100 120
Dermatan Sulfate, μM
 120

100
RATE s-1 X 103

80

60

40

20

0
0 5 10 15 20 25 30 35
Dermatan Disulfate, μM
GAG solution Standard Osmotic Stress (0.5 atm)

CSE in: K1/2, (µΜ) Vsat (s-1 x 103) K1/2, (µΜ) Vsat (s-1 x 103)

0.15 N, NaCl 3.3 ± 1.9 5.3 ± 1.3 4.3 ± 1.6 16 ± 3

0.15 N, NaCl, 2mM CaCl2 2.7 ± 0.8 5.7 ± 0.7 2.9 ± 0.4 20 ± 1

0.075 N NaCl 4.3 ± 2.8 6.1 ± 1.8 5.2 ± 2.3 15 ± 3

0.075 N NaCl,2mM CaCl2 1.3 ± 0.4 13.8 ± 0.1 0.34 ± .05 44 ± 1

0.15 N NaCl, 5mM CaCl2 4.8 ± 0.8 13.2 ± 0.1 2.7 ± 0.4 34 ± 2

HS in:

0.15 N, NaCl 4.0 ± 0.6 48 ± 4 5.5 ± 0.9 104 ± 10

15 N, NaCl, 2mM CaCl2 6.9 ± 0.9 97 ± 5 6.3 ± 0.4 189 ± 5

.075 N NaCl 6.6 ± 0.8 78 ± 4 4.8 ± 0.3 119 ± 3

.075 N NaCl, 2mM CaCl2 9.2 ± 1.8 204 ± 19.4 5.1 ± 0.8 283 ± 17
GAG solution Standard Osmotic Stress (0.5 atm)

DS in: K1/2, (µΜ) Vsat (s-1 x 103) K1/2, (µΜ) Vsat (s-1 x 103)

0.15 N, NaCl 74 ± 7 84 ± 5 37 ± 6 120 ± 8

0.15 N, NaCl, 2mM CaCl2 17 ± 3 97 ± 6 16 ± 1 136 ± 5

0.075 N NaCl 62 ± 4 125 ± 5 26 ± 2 136 ± 5

0.075 N NaCl,2mM CaCl2 19 ± 6 163 ± 21 3±1 116 ± 8

DDS in:

0.15 N, NaCl 17 ± 3 56 ± 5 9.4 ± 2 104 ± 10

15 N, NaCl, 2mM CaCl2 5.1 ± 0.4 73 ± 2 1.7 ± 0.3 106 ± 5

.075 N NaCl 7.5 ± 0.6 56 ± 2 3.8 ± 0.2 97 ± 2

.075 N NaCl, 2mM CaCl2 2.1 ± 0.2 73 ± 2.5 0.4 ± 0.8 107 ± 1
 TABLE 3

Disaccharide Structure and Anticoagulant Function.

Increased Efficiency with Osmotic Stress and Calcium

Hexuronic Acid Hexosamine sulfation pattern Efficiency M-1 s-1

Dermatan Sulfate Iduronic C4 4 X 104 (34)
Dermatan Disulfate Iduronic C4, C6 2 X 105 (81)
Chondroitin Sulfate E Glucuronic C4, C6 1 X 105 (81)
Heparan Sulfate Iduronic C2, N (variable) 5 X 104 (5)

Efficiency was estimated as the ratio between Vsat and K1/2 determined in titrations of antithrombin activity,
under an osmotic stress of 0.5 atm, with the indicated glycosaminoglycans in TRIS buffer pH 7.4, 0.075 N
NaCl and 2mM CaCl2 The increases in efficiency relative to reactions in standard TRIS buffer pH 7.4,
0.15N NaCl are indicated in parentheses.
GLYCOSAMINOGLYCANS REGULATE REACTIONS IN BIOGELS
Qui alium sequitur, nihil invenit, immo nee quaerit.
                                                                                  SENECA

(“He who follows another not only discover nothing, but is not even 
investigating.” Translation by RG Gummere)