We have identified two distinct mechanisms initiating the adhesion of flowing platelets to thrombogenic surfaces. The intergrin alpha IIb beta 3 promotes immediate arrest onto fibrinogen but is fully efficient only at wall shear rates below 600-900 s-1, perhaps because of a relatively slow rate of bond formation or low resistance to tensile stress. In contrast, glycoprotein Ib alpha binding to immobilized von Willebrand factor (vWF) appears to have fast association and dissociation rates as well as high resistance to tensile stress, supporting slow movement of platelets in continuous contact with the surface even at shear rates in excess of 6000 s-1. This eventually allows activated alpha IIb beta 3 to arrest platelets onto vWF under conditions not permissive of direct binding to fibrinogen. The coupling of these different functions may be crucial for thrombogenesis.

Platelet aggregation contributes to arresting bleeding at wound sites, but may cause occlusion of atherosclerotic vessels, thus curtailing blood flow to vital organs. According to current dogma, the integrin alphaIIbbeta3 plays an exclusive role in linking platelets to one another through interactions with fibrinogen or vWf. We demonstrate here that, depending on shearing flow conditions, this process may require vWf binding to glycoprotein Ibalpha, even when alphaIIbbeta3 is competent to bind adhesive ligands. Platelet activation induced solely by high shear stress is initiated by glycoprotein Ibalpha interaction with vWf, but results in aggregation only if the latter can bind concurrently to alphaIIbbeta3. In contrast, platelets exposed to high shear rate after activation by exogenous agonists such as ADP and epinephrine can aggregate when fibrinogen is the alphaIIbbeta3 adhesive ligand, yet only if vWf binding to glycoprotein Ibalpha can also occur. Thus, the latter interaction appears to provide a bond with biomechanical properties necessary to overcome the effects of high shear rate and initiate interplatelet cohesion. These findings highlight the distinct function of two adhesive receptors mediating platelet aggregation under varying fluid dynamic conditions, and modify the current interpretation of a crucial event in hemostasis and thrombosis.

Mechanisms mediating tumor cell attachment to the vessel wall under flow conditions are largely unknown. Therefore we analyzed the ability of human melanoma cells to adhere to an immobilized matrix during blood flow and determined the role of platelets in this process. In a parallel plate flow chamber, M21 melanoma cells were suspended in human blood and perfused over a collagen I matrix at a wall shear rate of 50 s ؊1 (2 dynes/ cm 2 ) to simulate venous flow over a thrombogenic surface. Melanoma cell interaction with the matrix or blood cells and platelets was monitored and quantified by fluorescence and confocal laser microscopy. Despite their ability to adhere to collagen I under static conditions, M21 cells failed to attach directly to this matrix during blood flow. However, they associated with adherent thrombi, and this resulted in stable melanoma cell arrest. Inhibition of platelet activation or platelet integrin ␣IIb␤3 function abolished M21 cell attachment. Melanoma cell interaction with thrombi was specific and required ␤3 integrin expression. M21-L cells which lack integrin ␣v␤3 failed to associate with thrombi and to arrest during blood flow. Transfection of these cells with the integrin subunits ␣v or ␣IIb resulted in variants expressing ␣v␤3, as in the wild type, or ␣IIb␤3. Both variants were able to associate with thrombi and to arrest during blood flow. Therefore, ␤3 integrin-mediated binding to activated platelets represents an efficient mechanism for melanoma cell arrest under flow, and this may contribute to the role of platelets in hematogenous metastasis.

Aggregation of blood platelets contributes to the arrest of bleeding at sites of vascular injury, but it can occlude atherosclerotic arteries and precipitate diseases such as myocardial infarction. The bonds that link platelets under flow conditions were identified using confocal videomicroscopy in real time. Glycoprotein (GP) Ib and von Willebrand factor (vWF) acted in synergy with IIbβ3 and fibrinogen to sustain platelet accrual at the apex of thrombi where three-dimensional growth resulted in increasing shear rates. The specific function of distinct adhesion pathways in response to changing hemodynamic conditions helps to explain hemostatic and thrombotic processes.

We have examined the interaction between the amino-terminal domain of platelet glycoprotein (GP) Ib␣ and immobilized von Willebrand Factor (vWF) under f low conditions in the absence of other components of the GP Ib-IX-V complex. Latex beads were coated with a recombinant fragment containing GP Ib␣ residues 1-302, either with normal sequence or with the single G233V substitution that causes enhanced affinity for plasma vWF in platelet-type pseudo-von-Willebrand disease. Beads coated with native fragment adhered to vWF in a manner comparable to platelets, showing surface translocation that ref lected the transient nature of the bonds formed. Thus, the GP Ib␣ extracellular domain is necessary and sufficient for interacting with vWF under high shear stress. Beads coated with the mutated fragment became tethered to vWF in greater number and had lower velocity of translocation than beads coated with the normal counterpart, suggesting that the G233V mutation lowers the rate of bond dissociation. Our findings define an approach for studying the biomechanical properties of the GP Ib␣-vWF bond and suggest that this interaction is tightly regulated to allow rapid binding at sites of vascular injury, while permitting the concurrent presence of receptor and ligand in the circulation.Platelet adhesion to von Willebrand factor (vWF) immobilized at sites of vascular injury initiates thrombus formation in areas of rapid blood flow (1) and represents a critical mechanism in hemostasis and thrombosis. The process is mediated by the glycoprotein (GP) Ib-IX-V complex, in which the aminoterminal domain of the GP Ib␣-chain (2, 3) contains the binding site for the vWF A1 domain (4, 5). This interaction supports platelet tethering to surfaces even at extremely high shear rates but without irreversible attachment. If no other bonds are formed, tethered platelets translocate in the direction of flow, albeit at a markedly lower velocity than freely flowing blood cells (6). On reactive substrates, however, initial contact allows the rapid establishment of additional bonds typically mediated by receptors of the integrin superfamily and results in essentially instantaneous irreversible adhesion, followed by events leading to subsequent thrombus development (1). At present, it is unknown whether any single domain of the GP Ib-IX-V complex can exhibit the vWF binding function of the intact receptor expressed on the platelet surface. Information in this regard could indicate whether linkage to the cytoskeleton (7,8) and͞or interactions between components of the complex (9, 10) contribute to vWF binding.We have addressed these questions by using a recombinant fragment comprising GP Ib␣ residues 1-302 (11). The isolated domain has been shown to undergo tyrosine sulfation and support modulator-dependent vWF binding as the native receptor (12). In the present studies, plastic beads were coated with the fragment, and its activity was tested in a flow field to mimic the function of a surface-expressed cell membrane receptor during vascular in...