Antiplatelet Agents Inhibit Platelet Adhesion and Aggregation on Glass Surface Under Physiological Flow Conditions: Toward a Microfluidic Platelet Functional Assay Without Additional Adhesion Protein Modification.

ABSTRACT: As the pathogenesis of arterial thrombosis often includes platelet adhesion and aggregation, antiplatelet agents are commonly used to prevent thromboembolic events. Here, a new microfluidic method without additional adhesion protein modification was developed to quantify the inhibitory effect of antiplatelet drugs on the adhesion and aggregation behavior of platelets on glass surfaces under physiological flow conditions. Polydimethylsiloxane-glass microfluidic chips were fabricated by soft photolithography. Blood samples from healthy volunteers or patients before and after taking antiplatelet drugs flowed through the microchannels at wall shear rates of 300 and 1500 second -1 , respectively. The time to reach 2.5% platelet aggregation surface coverage (Ti), surface coverage (A 150s ), and mean fluorescence intensity (F 150s ) were used as quantitative indicators. Aspirin (80 µM) prolonged Ti and reduced F 150s . Alprostadil, ticagrelor, eptifibatide, and tirofiban prolonged Ti and reduced A 150s and F 150s in a concentration-dependent manner, whereas high concentrations of alprostadil did not completely inhibit platelet aggregation. Aspirin combined with ticagrelor synergistically inhibited platelet adhesion and aggregation; GPIb-IX-von Willebrand factor inhibitors partially inhibited platelet aggregation, and the inhibition was more pronounced at 1500 than at 300 second -1 . Patient administration of aspirin or (and) clopidogrel inhibited platelet adhesion and aggregation on the glass surface under flow conditions. This technology is capable of distinguishing the pharmacological effects of various antiplatelet drugs on inhibition of platelet adhesion aggregation on glass surface under physiological flow conditions, which providing a new way to develop microfluidic platelet function detection method without additional adhesive protein modification for determining the inhibitory effects of antiplatelet drugs in the clinical setting.

Posttranslational modifications of platelet adhesion receptors.

Platelets play a key role in normal hemostasis, whereas pathological platelet adhesion is involved in various cardiovascular events. The underlying cause in cardiovascular events involves plaque rupture leading to subsequent platelet adhesion, activation, release, and eventual thrombosis. Traditional antithrombotic drugs often target the signal transduction process of platelet adhesion receptors by influencing the synthesis of some key molecules, and their effects are limited. Posttranslational modifications (PTMs) of platelet adhesion receptors increase the functional diversity of the receptors and affect platelet physiological and pathological processes. Antithrombotic drugs targeting PTMs of platelet adhesion receptors may represent a new therapeutic idea. In this review, various PTMs, including phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, lipidation, and proteolysis, of three platelet adhesion receptors, glycoprotein Ib-IX-V (GPIb-IX-V), glycoprotein VI (GPVI), and integrin αIIbß3, are reviewed. It is important to comprehensively understand the PTMs process of platelet adhesion receptors.

Pharmacologic inhibition of platelet vWF-GPIb alpha interaction prevents coronary artery thrombosis.

Under high shear arterial blood flow von Willebrand Factor (vWF) binds the platelet receptor glycoprotein (GP) Ibalpha, leading to platelet adhesion, activation and thrombosis. Blockade of vWF-GPIb alpha interactions by GPG-290 was investigated in a canine model of coronary artery thrombosis alone and in combination with clopidogrel. GPG-290 (100 microg/kg, n=6; 500 microg/kg, n=6) prolonged time to thrombotic occlusion (TTO) to 105+/-34 and 156+/-23 (p<0.05) min, respectively compared to the saline treated control group (32+/-6 min, n=6). Patency of the injured vessel was sustained in 1/6 (100 microg/kg) and 3/6 vessels (500 microg/kg) 4 hours after injury, in contrast to 0/6 in the control group. There was an increase in bleeding after the 500 microg/kg dose, but only at the 1 hr time point. Clopidogrel was studied in two dosing regimens representing either a clinical pretreatment regimen (PTR) of 4.3 mg/kg on day -2 followed by 1.1 mg/kg daily for 2 days prior to the procedure or pre-procedural loading dose regimen (LDR) of 4.3 mg/kg 3 hr pre-procedure. The PTR and LDR clopidogrel treatments prolonged TTO to 98.2+/-30.0 min and 136.1+/-39.5 min (p<0.05), and sustained patency in 1/6 and 4/8 vessels, respectively. However, template bleeding time in the LDR clopidogrel group was sustained higher than the control group. The combination of PTR clopidogrel and GPG-290 (100 microg/kg) prolonged TTO equivalent to LDR clopidogrel alone (141.4 +/- 35.1 min) and sustained patency in 3/7 dogs, without increased bleeding while LDR clopidogrel combined with 100 microg/kg GPG-290 prevented occlusion in 5/8 dogs and further prolonged TTO (173.5+/-32.6 min) but was associated with increased bleeding compared to control. GPG-290 is an antithrombotic agent that may be combined with lower doses of clopidogrel to yield similar antithrombotic efficacy as higher loading doses.

Drospirenone enhances GPIb-IX-V-mediated platelet activation.

BACKGROUND: Epidemiologic studies recently revealed that using drospirenone (DRSP)-containing contraceptives is associated with an increased risk of thrombosis in women. However, the underlying causality is unclear. OBJECTIVE: To study the effects of DRSP on coagulation in vitro and the probable mechanisms involved. METHODS: First, the effects of DRSP on the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were measured. Then, the effects of DRSP on platelet activation were investigated in response to low levels of collagen, adenosine 5'-diphosphate (ADP), thrombin, U46619, adrenaline and botrocetin/von Willebrand factor (VWF). RESULTS: DRSP has no direct effect on APTT, PT, TT, FIB and platelet aggregation induced by low levels of collagen, ADP, thrombin, U46619 or adrenaline. However, DRSP enhances botrocetin/VWF-induced platelet aggregation and VWF receptor glycoprotein Ib-IX-V (GPIb-IX-V)-mediated signaling. This enhancement can be blocked by the progesterone receptor membrane component 1 (PGRMC1) inhibitor AG205, or by the ADP scavenger apyrase and the cyclooxygenase inhibitor indomethacin. CONCLUSIONS: Although DRSP did not directly induce platelet activation, it obviously facilitated VWF receptor GPIb-IX-V-mediated platelet activation. The potential DRSP-binding protein PGRMC1 may play a role in this process. Our study also suggested that the inhibition of thromboxane A2 production and the activation of ADP receptors might prevent the side-effects of DRSP.

Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library.

Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbalpha binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies.

Ultrastructural analysis of megakaryocytes in GPV knockout mice.

Lesions in the genes for GPIb alpha, GPIb beta or GPIX result in a bleeding diathesis, the Bernard-Soulier syndrome (BSS), which associates a platelet adhesion defect with thrombocytopenia, giant platelets and abnormal megakaryocytes (MK). The role of GPV, also absent in BSS, was recently addressed by gene targeting in mice. While a negative modulator function for GPV on thrombin-induced platelet responses was found in one model, the absence of GP V had no effect on GPIb-IX expression or platelet adhesion. Our study extends previous results and reports that electron microscopy of bone marrow from the GPV knockout mice revealed a normal MK ultrastructure and development of the demarcation membrane system (DMS). There was a usual presence of MK fragments in the bone marrow vascular sinus. Immunogold labelling of MK from the knockout mice showed a normal distribution of GPIb-IX in the DMS and on the cell surface. The distribution of fibrinogen, vWF and P-selectin was unchanged with, interestingly, P-selectin also localised within the DMS in both situations. Thus GPV is not crucial to MK development and platelet production, consistent with the fact that no mutation in the GPV gene has as yet been described in BSS.

Antiphospholipid antibodies and the endothelium.

The interaction between aPL (particularly anti-beta 2GPI antibodies) and endothelium does represent a potential pathogenetic mechanism for the thrombotic manifestations of the syndrome. The autoantibody-mediated EC activation probably plays a role in sustaining the appearance of a proadhesive, proinflammatory, and procoagulant phenotype. The heterogeneity of the APS clinical manifestations is likely linked to the varied effects that aPL can induce on ECs and to the different functions that ECs display depending on the anatomic localization.

Antiphospholipid antibodies and the coagulation cascade.

Hemostasis is a highly controlled system of associated biophysical and biochemical events requiring a number of molecular and cellular interactions, among which molecular assembly at surfaces is an obligatory mechanism. The exposure of flowing blood to subendothelial components results in platelet adhesion, activation, and aggregation with simultaneous exposure of negatively charged phospholipids, which serves as a template for the formation of enzyme-cofactor-substrate complexes. The locally formed proteases activate surface-bound zymogens in a sequence culminating in the formation of thrombin. Fibrinogen is transformed into fibrin by thrombin, which may also activate protein C on phospholipid membranes when bound to TM. Activated protein C is a potent anticoagulant that inactivates coagulation-activated cofactors Va and VIIIa. During this process, proteins bound to the phospholipid surfaces may adopt new configurations and expose neoepitopes, which may elicit an immunologic response giving rise to the generation of antiphospholipid antibodies. These antibodies may then interfere with the procoagulant or anticoagulant activities of the target protein-phospholipid complexes. The apolipoprotein beta 2GPI and prothrombin are the most frequently found cofactors for antiphospholipid antibodies. Components of the protein C pathway have also been identified as cofactors. The pathophysiologic effects of antiphospholipid antibodies on the thrombotic accidents observed in patients with the antiphospholipid syndrome have not been established yet.

Glycoprotein Ib/IX/V binding to the membrane skeleton maintains shear-induced platelet aggregation.

The extracellular domain of glycoprotein (Gp) Ibalpha serves as the von Willebrand factor (vWf) receptor that triggers shear stress-dependent platelet aggregation. Its intracellular domain associates with actin-binding protein-280 (filamin 1a) that binds directly to filamentous actin, thereby linking the membrane skeleton to GpIbalpha. We examined the functional significance of GpIbalpha interactions with actin during platelet aggregation in response to 120 dyn/cm(2) shear stress. Lysates of resting and sheared platelets were centrifuged at approximately 13,000xg for 15 min, and GpIbalpha was immunoprecipitated from the lysate supernatant. GpIbalpha and coimmunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies specific for GpIbalpha and actin. We observed a significant increase in the amounts of actin coimmunoprecipitating with GpIbalpha as platelets aggregated in response to shear stress. Actin/GpIbalpha interactions reached a maximum after 90 s of shear stress. Monoclonal antibody (mAb) blockade of vWf binding to GpIbalpha inhibited shear stress-induced platelet aggregation and actin associating with GpIbalpha. Pretreatment of platelets with cytochalasin D resulted in the inhibition of actin binding to GpIbalpha in sheared platelets and in an increase in the rate and magnitude of platelet disaggregation. These data indicate that shear stress causes changes in the association between GpIbalpha and the actin-based membrane skeleton. The increased interaction between GpIbalpha and the actin-based membrane skeleton results from shear-induced vWf binding to GpIbalpha and is mechanoprotective in that it maintains shear-induced aggregation of activated platelets.

Anti-glycoprotein Ib causes platelet aggregation: different effects of blocking glycoprotein Ib and glycoprotein IIb/IIIa in the high shear filterometer.

In 1987 we reported that when blood was forced through a fine filter under pressure in the filterometer the platelets aggregated and blocked the filter. von Willebrand factor (vWF) and glycoprotein (Gp) IIb/IIIa and calcium were involved. Results with anti-GpIb were equivocal. We now report that all the anti-GpIb antibodies studied, glycocalicin, as well as some concentrations of aurin tricarboxylic acid caused platelet aggregation in the pre-filter blood and therefore could not be used in the filterometer. Using two different molecules that prevent vWF binding to GpIb and two anti-GpIIb/IIIa antibodies at two pressures it has now been shown that GpIb, vWf and high shear are primarily responsible for platelet retention at 0-5 s. Progressive platelet retention studied between 20 and 40 s required high shear and GpIIb/IIIa after the calcium influx mediated by GpIb/vWF binding. When GpIb was inhibited, GpIIb/Ila could not function normally, so GpIb inhibition resulted in decreased aggregation both at 0-5 s and at 20-40 s. Anti-GpIIlb/IIIa caused a minimal decrease in retention at 0-5 s and marked inhibition at 20-40 s. These findings fit and amplify concepts derived from other high shear methodologies. A diagram is presented of the events leading up to the final 'passivation' of the 'thrombus' in the filter when the surface of the aggregated platelets becomes unattractive.

Histamine reduces GPIbα-mediated adhesion of platelets to TNF-α-activated vascular endothelium.

Histamine and tumor necrosis factor-α (TNF-α) are critical mediators of acute and chronic inflammation that are generated by mast cells and macrophages in atherosclerotic lesions or systemically during allergic attacks. Both of them induce activation of vascular endothelium and thus may play a role in thrombosis. Here we studied the interplay between histamine and TNF-α in glycoprotein (GP) Ibα-mediated platelet adhesion to cultured human vascular endothelial cells under static and shear flow conditions. The stimulation of endothelial cells with histamine or TNF-α increased the number of adherent or slow rolling GP Ibα-coated microbeads or washed human platelets. However, the application of histamine to endothelium pre-activated by TNF-α inhibited GP Ibα-mediated platelet adhesion. These effects were found to be associated with changes in the concentration of ultra large von Willebrand factor (ULVWF) strings anchored to endothelium. The results of this study indicate that histamine released during mast cell degranulation may cause or inhibit thrombosis, depending on whether it acts on resting endothelial cells or on cells pre-activated by other inflammatory stimuli.

New strategy of platelet substitutes for enhancing platelet aggregation at high shear rates: cooperative effects of a mixed system of fibrinogen gamma-chain dodecapeptide- or glycoprotein Ibalpha-conjugated latex beads under flow conditions.

To construct platelet substitutes that have hemostatic properties over a wide range of shear rates, we used fibrinogen gamma-chain carboxy-terminal sequence HHLGGAKQAGDV (H12), which recognizes activated platelets at low shear rates, and a recombinant water-soluble moiety of the platelet glycoprotein (rGPIbalpha), which recognizes von Willebrand factor at high shear rates. Three kinds of samples were prepared for this purpose: H12-conjugated latex beads (H12-latex beads), rGPIbalpha-latex beads, and H12/rGPIbalpha-latex beads. These samples were evaluated in thrombocytopenia-imitation blood at various flow conditions. Based on ADP-induced platelet aggregation studies, the H12-latex beads significantly enhanced platelet aggregation via H12 binding with GPIIb/IIIa activated on the surface of activated platelets, whereas the rGPIbalpha-latex beads did not support platelet aggregation. In the case of the H12/rGPIbalpha-latex beads, the function of H12 was suppressed by steric hindrance from the larger rGPIbalpha bound to the latex bead. A mixture of the H12-latex beads and the rGPIbalpha-latex beads adhered to a collagen surface over a wide range of shear rates. In particular, at high shear rates, a cooperative effect was observed in the enhancement of platelet thrombus formation compared with H12-latex beads or rGPIbalpha-latex beads alone. We propose that a mixed system of H12- and rGPIbalpha-conjugated nanoparticles is a more effective platelet substitute than each of the beads used alone and has enhanced platelet aggregation properties.

Glycoprotein Ib alpha peptides inhibit thrombin and SFLLRN-induced platelet aggregation.

The platelet glycoprotein Ib(alpha) (GPIb(alpha)) receptor contains a high-affinity binding site for thrombin that, when occupied, augments platelet activation and aggregation in part via the 7-transmembrane domain receptor (7-TMDR). We have constructed a series of peptides derived from GPIb(alpha) that encompass the amino acid sequence F216-T240. We have studied the effect(s) of these peptides on platelet aggregation induced by thrombin or by the 7-TMDR peptide SFLLRN. Twenty-four peptides were synthesized from the peptide sequence F216-T240. Several of the peptides derived from the sequence W219-V227 of GPIb(alpha) inhibited platelet aggregation, which was primarily dependent on the presence of the amino acid sequence A224-N226 (AEN). These data suggest that a region within the GPIb(alpha) chain modulates the platelet aggregation induced by alpha-thrombin. These GPIb(alpha) peptides did not interfere with platelet aggregation induced by other agonists--for example, collagen, ristocetin, calcium ionophore, or botrocetin--which indicates that these GPIb(alpha) peptide-platelet interaction(s) are specific. Our studies provide another potential mechanism for modulating platelet activation and aggregation via synthetic and natural peptides.

Thrombopoietin combined with early-acting growth factors effectively expands human hematopoietic progenitor cells in vitro.

Thrombopoietin (TPO) is established as a powerful stimulant of megakaryocyte differentiation and platelet production both in vivo and in vitro. In preparation for future transplantation of ex vivo expanded CD34+ hematopoietic progenitor cells (HPCs), we have examined the in vitro effect of TPO on cultures of HPC when combined with other early-acting hematopoietic growth factors (GFs) in an attempt to decrease post-transplant thrombocytopenia and accelerate engraftment. By adding TPO to all possible combinations of GM-CSF, IL-3, and c-kit ligand (CKL) in a suspension culture system, we found a significant increase in both relative and absolute numbers of cells in cultures containing TPO of the megakaryocytic lineage and CD34+ cells after 14 days of culture. The most efficient GF combinations for expansion of cell populations of the megakaryocytic lineage and HPCs were TPO, GM-CSF, and CKL, which increased the number of cells of the megakaryocytic lineage 78 fold and the number of CD34+ cells 1.8 fold. The number of CD34+ cells decreased in the cultures containing GM-CSF and CKL with no TPO present, and the number of cells of the megakaryocytic lineage was increased merely 27 fold. Based on our findings, we suggest adding cells from HPCs expanded in cultures containing TPO, GM-CSF, and CKL to unexpanded stem cells for stem cell transplantation.

Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. (Blood. 2001;98:1038-1046)

Carbon monoxide modulates endotoxin-induced microvascular leukocyte adhesion through platelet-dependent mechanisms.

BACKGROUND: Although precise mechanisms remain to be determined, recent studies show that heme oxygenase-1 (HO-1), providing endogenous carbon monoxide (CO) and bilirubin, serves as an antiinflammatory enzyme. This study aimed to clarify roles of CO in regulation of microvascular adhesion of platelets and leukocytes in endotoxemia. METHODS: Rats pretreated with or without hemin were anesthetized with pentobarbital and received continuous infusion of endotoxin. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester and leukocyte behavior in mesenteric venules were visualized using intravital ultra-high-speed intensified fluorescence videomicroscopy. To examine the mechanisms for the effects of HO-1 on platelet and leukocyte behavior during endotoxemia, these studies were repeated with superfusion of either CO, bilirubin, or zinc protoporphyrine-IX. RESULTS: Endotoxin caused a marked depression of platelet velocity traversing along periendothelial regions, accompanied by augmented rolling and adhesion of leukocytes in venules. The endotoxin-elicited changes were attenuated by the HO-1 induction with hemin and restored by blockade of the enzyme activity with zinc protoporphyrine-IX, a potent inhibitor of HO-1. Such an inhibitory action of HO-1 on microvascular cell adhesion was reproduced by local superfusion of the buffer containing CO at micromolar concentrations. Such antiadhesive actions of CO on leukocytes disappeared under immunoneutralization of glycoprotein Ibalpha, an adhesion molecule against platelets, but not against leukocytes. Platelets isolated from hemin-treated rats increased their ability to generate CO and displayed lesser sensitivity of agonist-induced aggregation than those from controls. CONCLUSIONS: These results suggest that CO desensitizes endotoxin-induced adhesive responses of leukocytes, mainly through its ability to ameliorate platelet activation.

Glycoprotein Ib-mediated platelet activation. A signalling pathway triggered by thrombin.

Platelet activation by thrombin plays a major role in the development of haemostasis and thrombosis. Thrombin activates human platelets by cleaving the N-terminal region of G-protein-coupled protease-activated receptors (PARs). On the other hand, the platelet membrane glycoprotein GPIb acts as a thrombin-binding site and promotes platelet activation by low thrombin concentrations. We present here new evidence in favour of a thrombin receptor function for GPIb. We have selected conditions in which thrombin-GPIb interactions were enhanced by thrombin immobilization. Activation was studied independently of PAR cleavage by using active-site-blocked thrombin. We show that immobilized, proteolytically inactive thrombin induces platelet adhesion and spreading, dense granule secretion and integrin alphaIIbbeta3-dependent platelet-platelet interactions. The pathway must be dependent on GPIb because it is deficient in platelets from a patient with Bernard Soulier syndrome and inhibited by a monoclonal antibody to GPIb (SZ2) or by an excess of glycocalicin. Secreted ADP plays a major role in GPIb-dependent thrombin-induced platelet activation which is, in addition, regulated by cAMP concentration. Thrombin-induced GPIb-dependent platelet activation leads to tyrosyl phosphorylation of several proteins. Inhibition of platelet-platelet interactions and protein tyrosine phosphorylations by inhibitors of phosphatidylinositol 3-kinases and protein kinase C implies that activation of the latter are important steps of the GPIb-coupled signalling pathway triggered by thrombin.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity.

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

Role of Fc receptor gamma-chain in platelet glycoprotein Ib-mediated signaling.

Interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in human platelets. This study found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fc receptor gamma-chain (FcR gamma-chain), Syk, linker for activation of T cells (LAT), and phospholipase C gamma2 (PLCgamma2). Pretreatment of platelets with 10 microM PP1 completely inhibited these tyrosine phosphorylation events. On GPIb stimulation, Src and Lyn formed a complex with FcR gamma-chain and Syk, suggesting that Src and Lyn are involved in FcR gamma-chain tyrosine phosphorylation and downstream signals. In spite of the PLCgamma2 tyrosine phosphorylation, however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production. In Brij 35 lysates, FcR gamma-chain was found to constitutively associate with GPIb. The number of GPIb expressed on FcR gamma-chain-deficient platelets was comparable to that of the wild-type, as assessed by flow cytometry. However, tyrosine phosphorylation of Syk, LAT, and PLCgamma2 in response to vWF plus botrocetin was significantly suppressed, suggesting that FcR gamma-chain mediates activation signals related to GPIb. Compared with the aggregation response of wild-type platelets, that of FcR gamma-chain-deficient platelets in response to vWF plus botrocetin was impaired, implying that FcR gamma-chain is required for the full activation of platelets mediated by GPIb. (Blood. 2001;97:3836-3845)