Molecular basis of platelet activation by an αIIbβ3-CHAMPS peptide

Summary.  Background:  A novel method, known as computed helical anti-membrane protein (CHAMP), for the design of peptides that bind with high affinity and selectivity to transmembrane helices was recently described and illustrated using peptides that bind αIIb- and αv-integrin subunits, which induce selective activation of integrins αIIbβ3 and αvβ3, respectively [ 1 ]. Objectives:  In the present study, we have investigated the ability of an αIIb-CHAMPS peptide (termed integrin-activatory-peptide or IAP) to stimulate protein tyrosine phosphorylation and aggregation in human and mouse platelets. Methods:  The ability of IAP to stimulate platelet aggregation and dense granule secretion was measured in washed preparations of human and mouse platelets. Samples were taken for measurement of tyrosine phosphorylation. Results:  IAP stimulates robust tyrosine phosphorylation of the tyrosine kinase Syk and the FcR γ-chain, but only weak phosphorylation of PLCγ2. Aggregation to low but not high concentrations of IAP is reduced in the presence of the Src kinase inhibitor, PP1, or by inhibitors of the two feedback agonists, ADP and thromboxane A₂ (TxA₂) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA₂. Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin αIIbβ3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. Conclusions:  The present study demonstrates that the αIIb-CHAMPS peptide induces platelet activation through integrin αIIbβ3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR γ-chain and Syk. The use of the αIIb-CHAMPS peptide to study integrin αIIbβ3 function is compromised by non-integrin-mediated effects.     

Proteomic analysis of integrin αIIbβ3 outside-in signaling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions

Summary.  Background and Objectives : Outside-in integrin αIIbβ3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of αIIbβ3, and explore their role in platelet signaling. Methods and Results : Utilizing proteomics to search for novel platelet proteins that contribute to outside-in signaling by the integrin αIIbβ3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation-based signaling complex downstream of αIIbβ3. The proteins identified include the novel immunoreceptors G6f and G6b-B, and two members of the Dok family of adapters, Dok-1 and Dok-3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok-3 was also inducibly phosphorylated in response to the GPVI-specific agonist collagen-related peptide (CRP) and the PAR-1 and -4 agonist thrombin, independently of the integrin αIIbβ3. Tyrosine phosphorylation of Dok-1 and Dok-3 was primarily Src kinase-independent downstream of the integrin, whereas it was Src kinase-dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb-2 and SHIP-1 in fibrinogen-spread platelets. Conclusions : This study provides new insights into the molecular mechanism regulating αIIbβ3-mediated platelet spreading on fibrinogen. The novel platelet adapter Dok-3 and the structurally related Dok-1 are tyrosine phosphorylated in an Src kinase-independent manner downstream of αIIbβ3 in human platelets, leading to an interaction with Grb2 and SHIP-1.     

CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside-in signaling through integrin αIIbβ3

Summary.  Background: Agonist-induced inside-out signaling activates platelet integrin α_IIbβ₃, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through α_IIbβ₃ regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating α_IIbβ₃ function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl₃-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin β₃ subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.     

Changes in plasma glucose and lactate evoked by α and β2-adrenoceptor stimulation in conscious fasted rabbits

In conscious fasted rabbits, the iv infusion of salbutamol (3 micrograms/kg per min) and clonidine (2 micrograms/kg per min) induced a blood glucose increase amenable to blockade, respectively by ICI 118551 (1 micrograms/kg per min) and idazoxan (20 micrograms/kg per min). Amidephrine (10 micrograms/kg per min) and salbutamol mediated an increase in plasma lactate which was attenuated by prazosin (50 micrograms/kg, sc) and ICI 118551 respectively. Clonidine did not alter basal plasma lactate. The iv infusion of adrenaline (0.3 micrograms/kg per min) evoked an increase in plasma lactate more sensitive to blockade by ICI 118551 than by prazosin. ICI 118551 also shortened the hyperglycaemic response to adrenaline, 3-Mercaptopicolinic acid (25 mg/kg) reduced salbutamol- and adrenaline-mediated hyperglycaemia and increased at the same time the lactate/glucose ratio. Our data show that plasma lactate levels may be regulated by alpha 1- and beta 2-excitatory adrenoceptor stimulation. However, only the increase in blood lactate derived from beta 2-adrenergic stimulation seems to contribute to the overall catecholamine-mediated hyperglycaemia.     

Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 requires the docking protein LAT but is largely independent of αIIb/β3

Summary.  Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with αIIb/β3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The αIIb/β3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of αIIb/β3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cγ2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new αIIb/β3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSP1.     

Synthesis and secretion of α2-macroglobulin by human hepatocytes in culture

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. The cells were incubated in serum-free medium or 10% FCS-medium supplemented with insulin, glucagon and dexamethasone, and kept in culture for more than 2 weeks. Seventy-five per cent of the medium was changed regularly and assayed for alpha 2-macroglobulin (alpha 2-M), pregnancy zone protein, alpha 1-antitrypsin and albumin by means of ELISA. Significant amounts of alpha 2-macroglobulin were present in all cultures. During incubation, alpha 2-M accumulated in the medium and the quantity of alpha 2-M released from the cells by far exceeded protein associated with hepatocytes prior to incubation. In 24 h 10(6) hepatocytes secreted 160.5 +/- 82.2 ng of alpha 2-M (mean +/- SD, n = 5). Cell-associated, as well as secreted alpha 2-M appeared to be on native form, as determined by immunoisolates from lysed cells and culture supernatants. Pregnancy zone protein was only detected in about 50% of the cultures and its rate of secretion was less than 2 ng 24 h-1 per 10(6) cells. In contrast, culture medium contained considerable quantities of alpha 1-antitrypsin and albumin. In 24 h, 10(6) hepatocytes released greater than 2 micrograms alpha 1-antitrypsin and greater than 5 micrograms albumin. The present study suggests the hepatocyte to be of major importance for the synthesis of intravascular alpha 2-M.     

Activation of integrin αIIbβ3 in the glycoprotein Ib-high population of a megakaryocytic cell line, CMK, by inside-out signaling

Summary. Affinity/avidity state of integrin α_IIbβ₃ is regulated by intracellular inside‐out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to α_IIbβ₃ expressed in these cells by cellular agonists has not been demonstrated. We have re‐examined agonist‐induced α_IIbβ₃ activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of α_IIbβ₃ was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that α_IIbβ₃ expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIb^high cells was <0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA‐reactive GPIb^high cells. Not only PMA but also a calcium ionophore, A23187, induced α_IIbβ₃ activation, and PMA and A23187 had an additive effect on α_IIbβ₃ activation. Ligand binding to the activated α_IIbβ₃ in the GPIb^high CMK cells is totally abolished by an α_IIbβ₃‐specific antagonist, and inhibited by wortmannin, cytochalasin‐D and prostaglandin E₁, and the effects of these inhibitors on α_IIbβ₃ activation in the GPIb^high CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin‐treated CMK cells express PKC‐α, ‐β, ‐δ and ‐θ, and suggested that PKC‐α and/or ‐β appear to be responsible for PMA‐induced activation of α_IIbβ₃ in CMK cells.     

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α2β1 activation

Summary. Background: The platelet α₂β₁ integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α₂β₁ function can be activated via inside‐out signaling, similar to the prototypical platelet integrin α_IIbβ₃. However, signaling molecules that regulate α₂β₁ activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates α_IIbβ₃ activation. Objectives: We hypothesized that Rap1b positively regulates α₂β₁ during agonist‐induced platelet activation. Methods: To test whether Rap1b activates α₂β₁ downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b^?/? or wild‐type mice with diverse agonists and measured α₂β₁ activation using fluorescein isothiocyanate‐labeled monomeric collagen. We also examined the role of Rap1b in outside‐in signaling pathways by analyzing adhesion and spreading of Rap1b^?/? or wild‐type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b‐mediated events. Results: Rap1b^?/? platelets displayed comparable ADP‐induced or thrombin‐induced α₂β₁ activation as wild‐type platelets, but reduced convulxin‐dependent α₂β₁ activation. Rap1b^?/? platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild‐type platelets. Rap1b^?/? platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Conclusions: Rap1b is required for maximal GPVI‐induced but not ADP‐induced activation of α₂β₁ in murine platelets.     

Differential regulation of adapter proteins Dok2 and Dok1 in platelets, leading to an association of Dok2 with integrin αIIbβ3

BACKGROUND: We previously demonstrated that Dok2 is rapidly phosphorylated on tyrosine residues in platelets in response to thrombin, the immunoreceptor tyrosine-based activation motif-coupled collagen receptor glycoprotein (GP) VI, and by integrin alphaIIbbeta3. OBJECTIVES AND METHODS: In this study we further delineate the regulation of phosphorylation of Dok2 and compare this to the related adapter Dok1. RESULTS: We demonstrate expression of Dok1 in platelets and the unexpected observation that the adapter protein undergoes tyrosine phosphorylation in response to thrombin but not to GPVI or integrin alphaIIbbeta3. Furthermore, Dok1 phosphorylation is transient, peaking at 30 s and returning to basal by 5 min, whereas Dok2 phosphorylation is delayed but sustained. Dok2 phosphorylation, but not that of Dok1, is inhibited by Src kinase inhibitors and by chelation of intracellular calcium. Further, phosphorylation of Dok2 by thrombin and integrin alphaIIbbeta3 in mouse platelets is independent of Syk and phospholipase Cgamma2. Additionally, Dok2 coimmunoprecipitates with integrin alphaIIbbeta3 downstream of Src kinases. CONCLUSIONS: These results demonstrate differential modes of regulation of Dok1 and Dok2 in platelets. Further, they raise the interesting possibility that Dok2 plays an important role in integrin outside-in signaling through a physical and functional interaction with integrin alphaIIbbeta3.     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

MMP-2 regulates human platelet activation by interacting with integrin αIIbβ3

Summary. Background: Human platelets contain matrix metalloproteinases (MMPs) that are secreted during platelet activation. Platelet MMPs have been implicated in the regulation of cellular activation and aggregation. Although the proaggregatory effect of MMP‐2 has been demonstrated, the functional mechanism is not clearly understood. Objectives: This work was carried out in order to elucidate the biochemical mechanism of MMP‐2‐associated platelet activation and aggregation. Methods: MMP‐2 binding to the platelet surface was analyzed by flow cytometry. The cell surface target of MMP‐2 was identified in thrombin receptor‐activating peptide‐stimulated platelets by immunoprecipitation, Western blotting and fluorescence microscopy. A recombinant hemopexin‐like domain was used to characterize the nature of MMP‐2 binding to the platelet surface. The functional significance of MMP‐2 in platelet activation was investigated by quantitative measurements of the activation markers P‐selectin (CD62P) and active α_IIbβ₃. The role of MMP‐2 in platelet aggregation was analyzed with an aggregometer. Results: ProMMP‐2 binds to integrin α_IIbβ₃ in stimulated platelets in which proMMP‐2 is converted into MMP‐2. Fibrinogen was able to replace the α_IIbβ₃‐bound MMP‐2. The molecular interaction of MMP‐2 and integrin α_IIbβ₃ was abrogated by the recombinant human hemopexin‐like domain of MMP‐2, leading to reduced cell surface expression of activation markers CD62P and active α_IIbβ₃, and resulting in suppressed platelet aggregation. Conclusion: This work clearly demonstrates that platelet activation and aggregation is regulated by MMP‐2 that specifically interacts with integrin α_IIbβ₃. The C‐terminal hemopexin‐like domain of MMP‐2 is an essential element for binding to α_IIbβ₃.     

Platelet activation by dimeric β2-glycoprotein I requires signaling via both glycoprotein Ibα and apolipoprotein E receptor 2'

Summary. Background: Dimerization of β₂‐glycoprotein I (β₂‐GPI) by autoantibodies is thought to trigger the clinical manifestations observed in the antiphospholipid syndrome. Arterial thrombosis, a frequently occurring clinical manifestation of the antiphospholipid syndrome, is a process in which platelets play a crucial role. Previous work has shown that binding of dimeric β₂‐GPI to the platelet receptors apolipoprotein E receptor 2′ (ApoER2′) and glycoprotein Ibα (GPIbα) mediates increased platelet activation in an in vitro thrombosis model. Objective: The individual roles of ApoER2′ and GPIbα in mediating platelet activation by dimeric β₂‐GPI has hitherto been unclear. In this study, we have determined the roles of either receptor in platelet activation by dimeric β₂‐GPI. Methods: Platelet activation by dimeric β₂‐GPI was studied under conditions of flow. Intracellular signaling induced by dimeric β₂‐GPI was subsequently analyzed by means of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis. Results: The increase in platelet deposition onto a fibronectin surface under conditions of flow by dimeric β₂‐GPI was completely abolished by inhibition of the interaction of dimeric β₂‐GPI with either GPIbα or ApoER2′. Upon platelet stimulation with dimeric β₂‐GPI, GPIbα translocated to the cytoskeleton via the scaffold protein 14‐3‐3ζ. Concomitantly, ApoER2′ dissociated from the adapter protein Disabled1, presumably through phosphorylation of the cytoplasmic tail. Inhibition of one process could not inhibit the other. Conclusion: We show that dimeric β₂‐GPI signals via two distinct pathways in platelets, both of which are required for platelet activation. Abrogation of either signal results in loss of activation.     

Platelet adhesion to dimeric β2-glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ibα and apolipoprotein E receptor 2''

BACKGROUND: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (beta2GPI). Dimerized beta2GPI binds to apolipoprotein E receptor 2' (apoER2') on platelets and increases platelet adhesion to collagen under conditions of flow. AIM: To investigate whether the interaction between dimerized beta2GPI and platelets is sufficiently strong to resist shear stresses. METHODS: We studied the interaction of platelets with immobilized dimerized beta2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. RESULTS: We found that dimerized beta2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized beta2GPI was completely inhibited by the addition of soluble forms of both apoER2' and GPIbalpha, and the addition of receptor-associated protein and the removal of GPIbalpha from the platelet surface. GPIbalpha co-precipitated with apoER2', suggesting the presence of complexes between GPIbalpha and apoER2' on platelet membranes. The interaction between GPIbalpha and dimeric beta2GPI was of intermediate affinity (Kd = 180 nM) and Zn2+, but not Ca2+-dependent. Deletion of domain V from dimeric beta2GPI strongly reduced its binding to both GPIbalpha and apoER2'. Antibodies that inhibit the binding of thrombin to GPIbalpha inhibited platelet adhesion to dimeric beta2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbalpha had no effect. Dimeric beta2GPI showed reduced binding to low-sulfated GPIbalpha compared to the fully sulfated form. CONCLUSION: We show that platelets adhere to dimeric beta2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbalpha and apoER2'. These receptors are present in a complex on the platelet surface.     

Critical role of ADP interaction with P2Y12 receptor in the maintenance of αIIbβ3 activation: association with Rap1B activation

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.     

Alpha- and gamma-interferon (IFNα, IFNγ) but not interleukin-1 (IL-1) modulate synthesis and secretion of β2-microglobulin by hepatocytes

Soluble serum beta 2-microglobulin has been thought to result from membrane shedding by activated T-lymphocytes. This hypothesis could explain the increase of beta 2-microglobulin serum levels during virally induced mononucleosis, but not elevated levels as observed in other virally induced and in malignant diseases. In this paper we demonstrate that beta 2-microglobulin is a true secretory protein, and that its synthesis in hepatocytes is modulated by IFNs but not by IL-1. While the 45,000 MW HLA antigen can be found only in cell lysates, beta 2-microglobulin is shown to be secreted also into the culture medium like other secretory proteins (e.g. albumin-factor B-complement C3). Furthermore, interferon alpha (IFN alpha) as well as interferon gamma (IFN gamma) directly stimulate, in a dose- and time-dependent manner, beta 2-microglobulin synthesis by human hepatoma cells (Mz-Hep-1 and PLC/PRF5) and murine hepatocyte primary cultures. The increase of beta 2-microglobulin production induced by interferons is demonstrated at both the protein and the RNA level, indicating that interferon acts at a pretranslational level. The interferon effect on beta 2-microglobulin synthesis is specific since synthesis of secretory proteins like complement C3 or albumin, and of a structural protein like actin, remains unchanged. In contrast to IFN, IL-1, the main mediator of acute phase response, does not change beta 2-M biosynthesis rate. These data indicate that (i) beta 2-microglobulin is a secretory protein, (ii) IFNs but not IL-1 can mediate increased beta 2-M serum levels, and (iii) the liver may be its primary source.     

A tripeptide mimetic of von Willebrand factor residues 981–983 enhances platelet adhesion to fibrinogen by signaling through integrin βIIbβ3

BACKGROUND: RGD is a major recognition sequence for ligands of platelet alpha(IIb)beta3. OBJECTIVE AND METHODS: To identify potential binding sites for alpha(IIb)beta3 apart from RGD, we screened phage display libraries by blocking the enrichment of RGD-containing phages with a GRGDS peptide and identified a novel integrin recognition tripeptide sequence, VPW. RESULTS: Platelets adhered to an immobilized cyclic VPW containing peptide in a alpha(IIb)beta3-dependent manner; platelets and alpha(IIb)beta3-expressing CHO cells adhered faster to immobilized alpha(IIb)beta3-ligands in the presence of soluble VPW. In platelets adhering to fibrinogen, VPW accelerated the activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. In alpha(IIb)beta3-expressing CHO cells, VPW induced a faster formation of stress fibers. Sequence alignment positioned VPW to V980-P981-W982 in the von Willebrand factor (vWf) A-3 domain. In blood from a vWf-deficient individual, VPW increased platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired adhesion to vWf deficient in A-3. CONCLUSION: These data reveal a VPW sequence that contributes to alpha(IIb)beta3 activation in in vitro experiments. Whether the V980-P981-W982 sequence in vWf shows similar properties under in vivo conditions remains to be established.     

A mechanism to safeguard platelet adhesion under high shear flow: von Willebrand factor–glycoprotein Ib and integrin α2β1–collagen interactions make complementary, collagen-type-specific contributions to adhesion

BACKGROUND: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen. OBJECTIVE: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion. METHODS: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed. RESULTS: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen. CONCLUSIONS: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.