Brian Savage

We have found that the form of glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) expressed on nonstimulated platelets is a functional receptor that mediates selective and irreversible adhesion to immobilized fibrinogen. This occurs even in the presence of the elevated intracellular cAMP levels induced by prostaglandin E1 or after inhibition of protein kinase C activity by sphingosine. In the absence of inhibitors, platelets adhering to fibrinogen through GP IIb-IIIa become fully activated and aggregate with one another. Immobilized von Willebrand factor (vWF), in contrast, is recognized by nonstimulated platelets through another receptor, GP Ib. This interaction leads to a change in the ligand recognition specificity of GP IIb-IIIa that can then bind to immobilized vWF and mediate irreversible platelet adhesion and aggregation; this process, however, is inhibited by elevated intracellular cAMP levels or blockade of protein kinase C activity. Therefore, GP Ib and GP IIb-IIIa induce platelet activation through the selective recognition of immobilized vWF and fibrinogen, respectively, in the absence of exogenous agonists. Moreover, "nonactivated" and "activated" GP IIb-IIIa exhibits distinctly different reactivity toward surface-bound vWF, and the functional switch can be induced by the binding of vWF to GP Ib. These findings demonstrate the modulation of platelet function by two different adhesion receptors, GP Ib and GP IIb-IIIa, as well as the distinct dual role of the latter as the necessary common mediator of irreversible adhesion and aggregation on both fibrinogen and vWF.

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.

Three allelic differences in the alpha2 gene are associated with expression levels of the alpha2beta1 integrin on the platelet surface. We have previously defined two linked silent polymorphisms in the alpha2 gene coding region at nucleotides 807 (C or T) and 873 (G or A). We have now identified one rarer nucleotide polymorphism in the coding region at nucleotide 837 (T or C) and four additional linked polymorphisms within the introns that flank these coding sequences. Moreover, we have determined that the alloantigenic Br polymorphism, which resides in a distal coding region at nucleotide 1648, is also linked to the 837 polymorphism. Thus, three alpha2 gene alleles, defined by eight nucleotide polymorphisms, have now been discovered. Allele 1 (807T/837T/873A/Brb) is associated with increased levels of alpha2beta1; allele 2 (807C/837T/873G/Brb) and allele 3 (807C/837C/873G/Bra) are each associated with lower levels of alpha2beta1. Finally, we also show here that the rate of platelet attachment to type I collagen in whole blood under conditions of high shear rate (1,500/s) is proportional to the density of alpha2beta1 receptors on the platelet surface. Thus, the density of platelet alpha2beta1 could have an important impact on platelet adhesion to collagen in whole blood and therefore on platelet function in vivo, contributing to an increased risk of thrombosis or to bleeding in relevant disease states.