von Willebrand's disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand's disease.

105

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca 2؉ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP 3 ) and that treating platelets with the IP 3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca 2؉ flux and platelet shape change. Normal shape change was observed in G␣ q ؊/؊ mouse platelets, excluding a role for PLC␤ isoforms in this process. However, decreased shape change and Ca 2؉ mobilization were observed in mice lacking PLC␥2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca 2؉ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca 2؉ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-Fc␥RIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcR␥ chain. Taken together, these studies define an important role for PLC␥2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.

Section: Methodsmentioning

confidence: 99%

Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization

Mangin

Yao

Goncalves

et al. 2003

105

“…Materials-Bovine vWf (BvWf) was purified as previously described (17,18). Full-length complementary DNA (cDNA) for GPIb␣, GPIb␤, and GPIX cloned into the mammalian expression vector pDX, and CHO cells coexpressing GPIb␤ and GPIX (CHO␤IX) were kindly provided by Dr. J. Lopez (Houston, TX).…”

Section: Methodsmentioning

confidence: 99%

Interaction between Platelet Glycoprotein Ibα and Filamin-1 Is Essential for Glycoprotein Ib/IX Receptor Anchorage at High Shear

Williamson¹,

Pikovski²,

Cranmer³

et al. 2002

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIb␣ and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIb␣ intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569 -579 of the GPIb␣ cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant ⌬557-568 and ⌬569 -579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIb␣ for interaction with filamin-1.

“…Preparation of Native Human von Willebrand Factor-Human von Willebrand factor (HvWf) was purified to homogeneity from plasma cryoprecipitate according to the method of Montgomery and Zimmerman (24).…”

Section: Methodsmentioning

confidence: 99%

RhoA Sustains Integrin αIIbβ3Adhesion Contacts under High Shear

Schoenwaelder

Hughan²,

Boniface

et al. 2002

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, ␣ IIb ␤ 3 . Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, U46619, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin ␣ IIb ␤ 3 , by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin ␣ IIb ␤ 3 -dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase (Y27632) had no effect on integrin ␣ IIb ␤ 3 activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin ␣ IIb ␤ 3 adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin ␣ IIb ␤ 3 adhesion contacts under conditions of high shear stress.Platelet adhesion and aggregation at sites of vascular injury is essential for the arrest of bleeding and for subsequent vessel wall repair. The ability of platelets to adhere under conditions of rapid blood flow requires the synergistic contribution of multiple receptor-ligand interactions, foremost of which involves the interaction between von Willebrand factor (vWf) 1 and the two major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin ␣ IIb ␤ 3 . The vWf⅐GP Ib/V/IX interaction is characterized by a rapid association rate that enables efficient platelet tethering to the injured vessel wall, whereas subsequent integrin ␣ IIb ␤ 3 engagement of vWf is important for promoting platelet arrest (1). The vWf⅐GP Ib/V/IX interaction is indispensable for normal platelet function, because it slows platelet movement at the vessel wall thereby enabling receptors with slower intrinsic binding kinetics, i.e. integrins, to engage adhesive ligands. A similar dual-step adhesion mechanism, involving selectins and ␤ 2 integrins, is employed by leukocytes to adhere to post-capillary venules at sites of inflammation (2-4).A key requirement of the adhesion contacts formed by platelets and leukocytes is their ability to resist the detaching effects of rapidly flowing blood. In the case of platelets, the shear forces operating on adhesive bonds can be extremely high, particularly at sites of arteri...