Discussion
Beyond their role in blood clotting and homeostasis, functions of
platelets revealed more recently include adhesion to leukocytes
resulting in release of leukocyte and platelet constituents that damage
vessel walls and lead to diseases from myocardial infarction [27] to
ischemic stroke [28]. A clinically important phenomenon, largely
unexplained at the molecular level, is the increased susceptibility of
platelets of diabetic patients to activation and aggregation [29]
which presumably contribute to widespread vascular damage [30].
Further, a role for platelets as carriers of amyloid β, thereby acting
as a circulating sink for this peptide in order to reduce its
concentration in the brain has been recognized [5] though the exact
receptor for the peptide remains
ill-defined.
Availability of anti-Gal- and ABG-containing triplets in blood seems
to be determined mostly by the synthesis and/or plasma availability of
these antibodies since AOP1 and AOP2 together have been found to engage
nearly 36% of plasma albumin (our unpublished observation).
Identification of platelet surface molecule primarily recognized by
antibodies in anti-Gal/ABG-AOP1/AOP2-albumin triplets is crucial to
understanding the contribution of the latter towards platelet function.
Total blocking by jacalin of triplet attachment to denuded platelets
indicated the O-glycoprotein nature of triplet receptor on platelet
surface. Further, the most jacalin-reactive O-glycosylated molecule
identified on platelet membrane being a subunit of estimated molecular
weight 116 kDa, which is close to the reported molecular weights of the
GPIIb subunit, GPIIb/IIIa emerged as the most probable receptor for the
triplets. The next well-known O-glycosylated platelet membrane protein,
GP1bα, has a reported molecular weight of 135 kDa or above [31] and
is far less numerous than GPIIb/IIIa which is expressed at a density of
nearly 80,000 molecules per platelet, considered the highest in any cell
known [21,23]. This could explain the absence of any detectable
jacalin-binding protein other than the 116 kDa subunit in western blots
of platelet membrane (Fig.5). Other evidences implicating GPIIb/IIIa in
triplet attachment are: a) the protection of native triplet-containing
platelets from spontaneous ADP-mediated aggregation even while all
platelet components and plasma factors are available, and b) blocking of
ADP-mediated aggregation of denuded platelets by pre-bound jacalin
which, for reasons cited above, should be binding predominantly to
GPIIb/IIIa, an essential intermediate in ADP-mediated activation and
aggregation ofplatelets [21-23]. Another evidence suggesting
mediation of GPIIb/IIIa in triplet adhesion to platelets is the complete
blocking of this adhesion by pre-incubation of platelets with fibrinogen
which binds to GPIIb/IIIa (Fig. 4b). Thus while direct evidence is yet
to come, GPIIb/IIIa is the most likely receptor for
anti-Gal/ABG-AOP1/AOP2-albumin triplets on platelets.
Results above on amyloid β binding to platelets also support the
involvement of GPIIb/IIIa in triplet attachment since GPIIb/IIIa-bound
amyloid β, which is not extractable from platelets using anti-Gal- or
ABG-specific sugars, can be formed only on denuded platelets, showing
that this O-glycoprotein receptor is engaged and inaccessible to amyloid
β on normal platelets until the triplets are removed. Platelets have
been suggested to play a protective role against Alzheimer’s disease by
binding to amyloid β, thereby
serving as the plasma sink for this peptide to limit its availability in
brain[5]. On the other hand platelets are also reported to act as
carriers of amyloid β to perivascular cells of brain under conditions of
vascular damage [26]. Though GPIIb/IIIa has been reported to be
receptor for amyloid β on platelets [26], the present results show
that this may be true only of denuded platelets or of isolated platelet
membranes used for binding studies and that in circulating normal
platelets anti-Gal/ABG-AOP1/AOP2-albumin triplets mediate amyloid β
binding. In further proof for this conclusion are reports that diabetes
is the most common predisposing factor for Alzheimer’s disease(AD)
[33,34]. Though glucose is nearly as efficient as cellobiose as
ligand for ABG (Fig.2a) it does not dissociate the latter’s triplet at
normoglycemic levels (~4.7 mM) [1]. However
concentrations of glucose many fold higher than normal are attained in
diabetes, are inhibitory to ABG [11] and could dissociate triplets
of ABG and thereby that of anti-Gal as suggested by results in
Fig.2a.Though the resulting denuded platelets are capable of capturing
amyloid β, presumably through the newly exposed cell surface
O-glycoprotein vacated by triplet antibodies, they are more aggregative
as shown above and hence less stable, resulting in substantial
attenuation of platelet-mediated amyloid β-arresting activity of the
individual. The report that AD patients’ blood showed 39.57% increase
in platelet aggregates and 53.3 % increase in leukocyte-platelet
complexes [35] also underlined the above conclusion. The myriad of
platelet-mediated vascular injuries accompanying diabetes also support
triplet-mediated protection of platelets. Thus platelet-dependent
thrombosis is proportional to blood glucose levels in coronary artery
disease [30]. Even short term hyperglycemia in diabetics is reported
to cause vascular occlusions through platelet activation [30,36].
Particularly, the cascade of inflammatory events leading to cerebral
amyloid angiopathy preceding AD had been reported to be triggered by
amyloid β binding to exposed GpIIb/IIIa on platelets that adhere to
vessel walls [26].
Platelet-leukocyte adhesion facilitated by platelet surface
GpIIb/IIIa is trigger for
synthesis and release of several inflammatory factors by leukocytes.
These factors have been implicated in acute myocardial infarction
[37] and stroke [28]. Since the protective cover for GpIIb/IIIa,
presumably provided by triplets, disappears during hyperglycemia and
renders the platelets susceptible to adhesion of leukocytes the results
above may explain in molecular terms the contribution of diabetes
towards GpIIb/IIIa-mediated pathophysiology of the above disorders. In
summary the present results reveal the molecular basis for triplet
anchoring on platelets, for the absence of platelet aggregation in
normal state and for platelets serving as amyloid β sink in circulation.
Data also offer possible reasons for diabetes-mediated platelet
vulnerability and platelet-leukocyte adhesion.