Susana Larrucea

Localization of the Adhesion Receptor Glycoprotein Ib-IX-V Complex to Lipid Rafts Is Required for Platelet Adhesion and Activation

The platelet glycoprotein (GP) Ib-IX-V complex mediates the attachment of platelets to the blood vessel wall by binding von Willebrand factor (VWF), an interaction that also transmits signals for platelet activation and aggregation. Because the complex is extensively palmitoylated, a modification known to target proteins to lipid rafts, we investigated the role of raft localization in GP Ib-IX-V functions. In unstimulated platelets, a minor portion of the complex localized to Triton-insoluble raft fractions; this portion increased three to sixfold with platelet activation by VWF. Raft-associated GP Ib-IX-V was selectively palmitoylated, with GP Ib-IX-V–associated palmitate increasing in the raft fraction on VWF-mediated activation. The raft fraction was also the site of association between GP Ib-IX-V and the Fc receptor FcγRIIA. The importance of this association was demonstrated by the ability of the FcγRIIA antibody IV.3 to inhibit shear-induced platelet aggregation. Disruption of rafts by depleting membrane cholesterol impaired several GP Ib-IX-V–dependent platelet fractions: aggregation to VWF under static conditions and under shear stress, tyrosine phosphorylation, and adhesion to a VWF surface. Partial restoration of membrane cholesterol content partially restored shear-induced platelet aggregation and tyrosine phosphorylation. Thus, localization of the GP Ib-IX-V complex within rafts is crucial for both platelet adhesion and postadhesion signaling.

Platelets of Female Mice Are Intrinsically More Sensitive to Agonists Than Are Platelets of Males

Objective—It has been reported that women fare worse after ischemic coronary events, but the mechanisms remain unclear. Because platelets play a central role in the formation of occlusive thrombi at sites of ruptured atherosclerotic plaques, we studied male/female paired mouse littermates for sex differences in platelet function. Methods and Results—We compared platelet reactivity in male/female mouse littermates by monitoring agonist-induced fibrinogen (FGN) binding and platelet aggregation. Compared with the platelets from males, platelets from females bound more FGN in response to low concentrations of thrombin and collagen-related peptide. Female platelets also demonstrated greater aggregation in response to adenosine diphosphate and collagen-related peptide. Platelet protein tyrosine phosphorylation on activation also showed small differences between sexes. These differences are independent of platelet size and surface expression of &agr;IIb&bgr;3 and GPIb-IX-V, and they were not blocked by apyrase or aspirin. The sex differences we observed were intrinsic to platelets, because they were observed in washed platelets, but not when platelets were in plasma. Conclusions—The platelets of female mice were more reactive than those of males in a manner independent of COX-1 and secreted ADP.

Release of podocalyxin into the extracellular space. Role of metalloproteinases

Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells (podocytes) of kidney glomeruli where it plays an important role in maintaining the plasma filtration. PODXL is also expressed in other types of cells but its function is ignored. A recombinant soluble fragment of the PODXL ectodomain modifies the signaling of the membrane bound PODXL. Based on this antecedent, we aimed at investigating whether PODXL could be cleaved and released into the extracellular space as a soluble peptide. In this study, we used a fusion protein of human PODXL and green fluorescent protein expressed in CHO cells (CHO-PODXL-GFP) and a human tumor cell (Tera-1) inherently expressing PODXL. PODXL was detected by wide-field microscopy in the Golgi, the plasma membrane and in a vesicular form preferentially located at the leading edges of the cell and also progressing along the filopodium. We detected PODXL in the insoluble and soluble fractions of the extracellular medium of CHO-PODXL-GFP cells. Stimulation of protein kinase C (PKC) by Phorbol-12-myristate-13-acetate (PMA) enhanced the release of PODXL to the extracellular space whereas this effect was prevented either by inhibitors of PKC or specific inhibitors of matrix metalloproteinases. It is concluded that intact PODXL is released to the extracellular space as a cargo of microvesicles and also as a soluble cleaved fragment of ectodomain.

A variant thrombasthenic phenotype associated with compound heterozygosity of integrin β3-subunit: (Met124Val)β3 alters the subunit dimerization rendering a decreased number of constitutive active αIibβ3 receptors

We report the analysis of a variant case of thrombasthenic phenotype that is a compound heterozygote for two mutations located within the metal ion dependent adhesion site (MIDAS) of the β3 subunit.The patient inherited a maternal allele carrying the Met124Val substitution and a paternal allele that changes Asp119 to Tyr. Phenotyping of the human platelet antigen 1 (HPA-1) showed that the platelet αIIbβ3 complex in the patient was mostly accounted for by the Asp 119Tyr allele that does not bind to fibrinogen (Fg). The patient showed agonistinduced binding of platelets to Fg but neither binding to PAC-1 nor cell aggregation could be detected, most likely due to the minute expression (≤5%) of αIIb(124Val)β3 receptors. CHO cells expressing (124Val)β3 showed a diminished surface expression of aIIbs3, enhanced adhesion to immobilized Fg, and spontaneous aggregation in the presence of soluble Fg, suggesting that (124Val)β3 may confer constitutive activity to the αIIb(124Val)β3 receptors. A distinct feature of these cells is the failure of DTT to enhance the binding to soluble Fg and the formation of cell aggregates. The substitution of (124Met)β3 by either a polar or a positively charged amino acid restored the surface exposure and function of the αIIbβ3 receptors whereas a negatively charged residue did not.

α-Adrenergic-mediated activation of human reconstituted fibrinogen receptor (integrin αIibβ3) in Chinese hamster ovary cells

This work reports the functional studies of CHO cells coexpressing a-adrenergic (aAR) and human fibrinogen (Fg) receptors (integrin αIibβ3). Stimulation of these cells with a-agonists produced a transient rise in the free cytosolic calcium (Ca++) accompanied by enhanced binding to soluble Fg, and these effects were prevented by specific aAR antagonists. The a- adrenergic-induced activation of αIibβ3 in CHO-αIibβ3-aAR increased the rate of adhesion and extension of cells onto Fg coated plates, and also induced a soluble Fg- and αLIIba3-dependent formation of cell aggregates, whereas no effects were observed by the stimulation of CHO-αIibβ3 cells.a-Adrenergic antagonists, the ligand mimetic peptide RGDS, pertussis toxin (PTX), or EDTA, they all prevented the a-adrenergic stimulation of adhesion and aggregation. However, inhibition of PKC prevented the a-adrenergic stimulation of cell adherence, whereas blocking the intracellular Ca++ mobilization impeded the stimulation of cell aggregation.The α-adrenergic activation was associated with phosphorylation of a protein of ~100 kDa and proteins of the MAPK family. The former was selectively phosphorylated by α-adrenergic stimulation whereas the latter were phosphorylated by the binding of cells to Fg and markedly intensified by a-adrenergic stimulation.