PTP-1B is an essential positive regulator of platelet integrin signaling

Outside-in integrin αIIbβ3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein–tyrosine phosphatase (PTP)–1B in this process. In resting platelets, c-Src forms a complex with αIIbβ3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to αIIbβ3 triggers PTP-1B recruitment to the αIIbβ3–c-Src–Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B–deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from αIIbβ3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B–deficient platelets are defective in outside-in αIIbβ3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in αIIbβ3 signaling in platelets

Dynamics of von Willebrand factor-mediated platelet aggregation in laminar flow : physical and molecular determinants

Von Willebrand factor (vWF) is a large multimeric protein found in plasma, intracellular stores of platelets and endothelial cells, as well as in the extracellular matrix. VWF has been implicated in venous and arterial thrombosis. Its importance in the primary adhesion of platelets at sites of vascular injury, and for platelet aggregation at very high shear rates was clearly demonstrated by other investigators. We have investigated the role of vWF in the recruitment of platelets activated with low concentrations of agonist, such as may be found with partial ADP secretion or thrombin generation in vivo, at physiologic shear rates (G) ranging from 100--2000 s--1.In the presence of ristocetin, soluble vWF bound to the glycoprotein Ib receptor (GPIb), and mediated shear-associated aggregation independently of the glycoprotein IIb-IIIa receptor (GPIIb-IIIa), with very few vWF monomer equivalents required to achieve high capture efficiencies (alphaG; reflecting initial rates of aggregation). Activation of washed platelets in the absence of soluble ligands, with low concentrations of the physiologic agonists, ADP or thrombin, resulted in good aggregation. Participation by GPIb was shearrate dependent, with the extent of contribution further varying with activation conditions. Inhibition of vWF-GPIb interactions in ADP and thrombin-induced aggregation, caused 25 and 50% inhibition of alpha G at G = 300 and 1000 s--1 respectively. alpha G's were similar for both agonists, and showed an absolute dependence on activated GPIIb-IIIa in each case. Further investigations using thrombin versus ADP as activator, however, revealed differences in participation by other surface-expressed ligands, in particular Fg. Thus, antibodies against TSP and Fg inhibited aggregation differentially, depending on shear rate and agonist activating conditions. Removal of catalytically-active thrombin from its receptors by antagonists hirudin and PPACK at ≥1 minute following activation, allowed normal secretion to occur from alpha-granules (monitored using P-selectin). However, the thrombin antagonists significantly decreased both platelet aggregation and surface-expression of vWF and TSP for platelets activated at low (0.05 U/ml), but not intermediate (0.2 U/ml) thrombin concentrations. In conclusion, all our studies demonstrated a consistently important cross-bridging role for vWF, but multi-ligand, multi-receptor participation was required for optimal shear-associated aggregation of platelet suspensions activated at very low agonist concentrations

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets

Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbβ3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbβ3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbβ3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbβ3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with alpha IIb beta 3 and LUBAC

Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P </p

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC.

Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P &lt; .01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P &lt; .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P &lt; .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells

ADAP is required for normal αIIbβ3 activation by VWF/GP Ib-IX-V and other agonists

Interaction between von Willebrand factor (VWF) and platelet GP Ib-IX-V is required for hemostasis, in part because intracellular signals from VWF/GP Ib-IX-V activate the ligand-binding function of integrin αIIbβ3. Because they also induce tyrosine phosphorylation of the ADAP adapter, we investigated ADAP's role in GP Ib-IX-V signal transduction. Fibrinogen or ligand-mimetic POW-2 Fab binding to αIIbβ3 was stimulated by adhesion of ADAP(+/+) murine platelets to dimeric VWF A1A2 but was significantly reduced in ADAP(−/−) platelets (P < .01). αIIbβ3 activation by ADP or a Par4 thrombin receptor agonist was also decreased in ADAP(−/−) platelets. ADAP stabilized the expression of another adapter, SKAP-HOM, via interaction with the latter's SH3 domain. However, no abnormalities in αIIbβ3 activation were observed in SKAP-HOM(−/−) platelets, which express normal ADAP levels, further implicating ADAP as a modulator of αIIbβ3 function. Under shear flow conditions over a combined surface of VWF A1A2 and fibronectin to test interactions involving GP Ib-IX-V and αIIbβ3, respectively, ADAP(−/−) platelets displayed reduced αIIbβ3-dependent stable adhesion. Furthermore, ADAP(−/−) mice demonstrated increased rebleeding from tail wounds. These studies establish ADAP as a component of inside-out signaling pathways that couple GP Ib-IX-V and other platelet agonist receptors to αIIbβ3 activation