Page 682 - Hematology_ Basic Principles and Practice ( PDFDrive )

574 Part V Red Blood Cells

In the physiologic form of the polymer, the component strings of 35
Hb molecules in a double strand are half-staggered and have a slight
twist, creating a fiber that is approximately 21 nM in diameter and
is composed of one central and six peripheral double strands. The 30
crystal formed in vitro lacks the twist, but its molecular structure is
known in great detail. 25
Hb solubility
Role of Hemoglobin S Solubility 20

The RBC’s hydration state dominates the physical-chemical behavior 15 HbF
HbA 2
of HbS. The solubility of deoxy-HbS (approximately 16 g/dL, HbA
measured under laboratory conditions) is much lower than the RBC HbC
mean cell Hb concentration (MCHC). So, even partial cellular 10
deoxygenation can raise deoxy-HbS concentration above its solubility 0 0.2 0.4 0.6 0.8
limit, allowing polymerization to occur. The biophysical effect of A Fraction Hb X
macromolecular crowding (boosting a protein’s activity far above that
predicted from concentration alone) confers nonideal behavior upon
cytoplasmic constituents, augmenting likelihood for polymerization 0.7
at any given degree of deoxygenation. SS
In vitro studies carried out under (nonphysiologic) equilibrium
conditions of stable oxygen tension and long-time scale corroborate
crystallographic identification of critical amino acids involved in
atomic contacts by revealing the influence of other Hbs on HbS solu-
3
bility (Fig. 41.4). When different Hbs are mixed together, the tetra- Polymer fraction 0.35 +
mers dissociate into dimers that intermix and randomly assemble in + +
a binomial distribution to reform tetramers. This clarifies the impact + + +
of naturally occurring, intracellular Hb mixtures. In mixtures of HbS AS + + + +
S
A
and HbA, overall solubility is improved because the hybrid αβ /αβ + +
S
S
tetramer integrates into polymer only one half as well as the αβ /αβ + +
tetramer (Fig. 41.4A). Addition of HbF to HbS has a greater sparing + + + + + + +
S
effect because neither the αγ/αγ nor the hybrid αβ /αγ tetramer can 0
be incorporated into polymer. In this regard, HbC has the same effect 0 50 100
as HbA, and HbA 2 has the same effect as HbF (see Fig. 41.4A). This B Oxygen saturation (%)
sparing effect of HbA is such that much lower Hb oxygen saturation
is required for polymer to form in HbAS than in HbSS RBCs Fig. 41.4 DEOXYHEMOGLOBIN S SOLUBILITY, DEFINED BY
(Fig. 41.4B). STUDIES UNDER EQUILIBRIUM CONDITIONS. (A) Admixture of
other hemoglobins with hemoglobin S raises overall solubility in absence of
oxygen. The x-axis indicates the proportion of admixed nonsickle Hb. (B)
Kinetics of Polymerization The hemoglobin oxygen saturation required to initiate intracellular polymer
formation (i.e., polymer fraction) is much lower for HbAS RBC than for
HbSS RBC. (A, Reproduced with permission from Poillon WN, Kim BC, Rodgers
Laboratory measurements of polymerization kinetics, enabled by
inducing (nonphysiologic) near-instantaneous and complete conver- GP, et al: Sparing effect of hemoglobin F and hemoglobin A 2 on the polymerization of
sion of HbS from R (oxy) to T (deoxy) state, reveal a delay until hemoglobin S at physiologic ligand saturations. Proc Natl Acad Sci U S A 90:5039,
4
polymer forms explosively. This inherent delay time is inversely 1993; B, reproduced with permission from Schechter AN, Noguchi CT: Sickle hemo-
related to an extremely high power of the initial Hb concentration; globin polymer: Structure-function correlates. In Embury SH, Hebbel RP, Mohandas
it is approximately 10 ms at Hb of 40 g/dL, but it is 100,000 seconds N, Steinberg MH, editors: Sickle cell disease: Basic principles and clinical practice,
at Hb 20 g/dL (Fig. 41.5A). HbS solutions and sickle RBCs behave New York, 1994, Raven Press.)
similarly in this regard. Delay times must vary enormously from cell
to cell because they are dominated by the marked heterogeneity in
MCHC (i.e., shorter delay for more dehydrated cells) and are influ- short delay times (Fig. 41.5D) reflect simultaneous formation of
enced by the presence of any non-S Hb (i.e., longer delay for presence multiple nucleation sites in cells that polymerize rapidly.
of HbA, C, or F) (Fig. 41.5E). Admixture of 20% to 30% HbA with
+
HbS (simulating HbS-β -thalassemia) increases the delay time
10 to 100 fold, and admixture of 20% to 30% HbF with HbS Polymerization Under (Patho)physiologic Conditions
3
4
increases it by 10 - to 10 -fold.
The mechanism of such polymer formation is hypothesized to In physiology, sickle RBCs are neither at equilibrium with constant
proceed by a two-step, double-nucleation process (Fig. 41.5F). oxygen tension nor undergoing instantaneous or complete deoxygen-
Accordingly, the initial homogeneous nucleation takes place in bulk ation. Rather, irrespective of the inherent delay time, the rate of
solution, during which small numbers of tetramers associate, with deoxy-HbS polymer growth in vivo is limited by the rate at which
accumulation not favored until a critical nucleus size develops (esti- RBC deoxygenation develops during microvascular passage. Since
mated to be 30 to 50 tetramers). Only then can new tetramers be this transit time is on the order of ~1 second, it probably effectively
added lengthwise to form a large polymer. After this occurs, hetero- renders irrelevant any inherent delay times of less than ~1 second
4
geneous nucleation causes explosive, autocatalytic polymer formation (Fig. 41.5G). Thus kinetic considerations argue that most RBCs in
as new fibers form and extend on the surface of the preexisting patients with sickle cell anemia are unlikely to sickle during their
polymer. It is the time until this explosive formation occurs that labo- passage through the microcirculation unless something, such as
ratory experiments detect as the inherent delay time. It is believed RBC–endothelial adhesion, slows their transit.
that the striking irreproducibility of long delay times (Fig. 41.5B) Predictability is complicated by the marked heterogeneity among
reflects stochastic formation of a single (or at least very few) homo- sickle RBCs in MCHC and HbF content, as well as the natural
geneous nucleation event(s) in cells that slowly polymerize and that biologic variability in capillary transit times. A good qualitative