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of these antibodies since AOP1 and AOP2 together have been found to engage nearly 36% of plasma albumin (our unpublished observation).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.