An acquired inhibitor to the GPVI platelet collagen receptor in a patient with lupus nephritis

Summary.  Background : GPVI is a major platelet collagen signaling receptor. In rare cases of immune thrombocytopenic purpura (ITP), autoantibodies to GPVI result in receptor shedding. Objectives : To investigate a possible pathogenic role of plasma anti-GPVI antibody located in a woman with lupus nephritis. Methods : Measured were (i) platelet aggregation to collagen and convulxin, (ii) platelet GPVI expression (flow cytometry and western blotting), (iii) plasma soluble GPVI (sGPVI, dual antibody ELISA), and (iv) plasma anti-GPVI antibody (ELISA using recombinant sGPVI). Results : In 2006 and early 2007, the patient had a normal platelet count but a virtual absence of platelet aggregation to collagen and convulxin. Her platelets responded normally to other agonists including cross-linking ITAM-dependent FcγRIIA by monoclonal antibody, IV.3. Flow cytometry and western blotting showed a platelet deficiency of GPVI. Plasma sGPVI levels were undetectable whereas ELISA confirmed the presence of anti-GPVI antibody. Sequencing revealed a normal GPVI cDNA structure. The patient's plasma and the isolated IgG3 fraction activated and induced GPVI shedding from normal platelets. A deteriorating clinical condition led to increasingly strict immunosuppressive therapy. This was globally associated with a fall in plasma anti-GPVI titres, the restoration of platelet GPVI and the convulxin response, and the loss of her nephrotic syndrome. Conclusions: Our results show that this patient acquired a potent anti-GPVI IgG3 antibody with loss of GPVI and collagen-related platelet function. Further studies are required to determine whether anti-GPVI antibodies occur in other lupus patients with nephritis.     

Compromised ITAM-based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background:  Receptors on platelets that contain immunoreceptor tyrosine-based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives:  We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results:  Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen-related peptide, or to FcγRII-activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti-GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa-blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full-length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10-kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL⁻¹ soluble GPVI by ELISA (normal plasma, ∼15 ng mL⁻¹) and IgG purified from patient plasma caused FcγRIIa-mediated, EDTA-sensitive cleavage of both GPVI and FcγRIIa on normal platelets. C onclusions:  In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.     

Human platelet glycoprotein VI function is antagonized by monoclonal antibody-derived Fab fragments

Summary.  Platelet interactions with adhesive ligands exposed at sites of vascular injury initiate the normal hemostatic response but may also lead to arterial thrombosis. Platelet membrane glycoprotein (GP)VI is a key receptor for collagen. Impairment of GPVI function in mice results in a long-term antithrombotic protection and prevents neointimal hyperplasia following arterial injury. On the other hand, GPVI deficiency in humans or mice does not result in serious bleeding tendencies. Blocking GPVI function may thus represent a new and safe antithrombotic approach, but no specific, potent anti-GPVI directed at the human receptor is yet available. Our aim was to produce accessible antagonists of human GPVI to evaluate the consequences of GPVI blockade. Amongst several monoclonal antibodies to the extracellular domain of human GPVI, one, 9O12.2, was selected for its capacity to disrupt the interaction of GPVI with collagen in a purified system and to prevent the adhesion of cells expressing recombinant GPVI to collagen and collagen-related peptides (CRP). While 9O12.2 IgGs induced platelet activation by a mechanism involving GPVI and FcγRIIA, 9O12.2 Fab fragments completely blocked collagen-induced platelet aggregation and secretion from 5 µg mL⁻¹ and fully prevented CRP-induced activation from 1.5 µg mL⁻¹. 9O12.2 Fabs also inhibited the procoagulant activity of collagen-stimulated platelets and platelet adhesion to collagen in static conditions. Furthermore, 9O12.2 Fabs impaired platelet adhesion, and prevented thrombi formation under arterial flow conditions. We thus describe here for the first time a functional monoclonal antibody to human GPVI and demonstrate its effect on collagen-induced platelet aggregation and procoagulant activity, and on thrombus growth.     

Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation.

We examined the basis of the differences observed between different collagen preparations in their ability to aggregate platelets and support their adhesion under flow. As in previous studies, we found fibrillar collagen to be 10-fold more potent than acid-soluble collagen in inducing platelet aggregation and found that acid-soluble collagen did not support the adhesion of washed platelets under flow. Further, platelets in whole blood adhered to surfaces coated with either fibrillar or acid-soluble collagen, but thrombi formed faster and grew larger on fibrillar collagen. As a possible basis for this difference, we found that fibrillar collagen, but not acid-soluble collagen, contains a substantial quantity of von Willebrand factor (VWF), as demonstrated by enzyme-linked immunosorbent assay and by the ability of fibrillar collagen to support the adhesion of VWF antibody-coated beads and to agglutinate GPIb-IX-V complex-expressing Chinese hamster ovary cells. Supporting a role for VWF in collagen-induced platelet aggregation, aggregation induced by acid-soluble collagen was greatly enhanced by added VWF. Further, platelet aggregation by fibrillar collagen was partially blocked by a GPIbalpha antibody that inhibits the GPIb-VWF interaction. Taken together, these results suggest that much of the difference in prothrombotic potency of different collagens is directly related to their differences in VWF content. This probably accounts for the different conclusions made regarding the relative importance of different direct and indirect collagen receptors in collagen-dependent platelet functions and further emphasizes the close synergistic roles of the GPIb-IX-V complex and the collagen receptors GPVI and alpha2beta1 in supporting platelet adhesion.     

A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functions

Summary.  Background and objectives:  Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets. Methods and results:  In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin α2β1 and αIIbβ3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu¹⁸⁰ to Asn²⁴⁹ as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE²⁰⁵/A²⁰⁶TA and NKV²¹⁸/F²¹⁹TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. Conclusions:  We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents.     

Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.

A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.     

血小板膜糖蛋白Ⅵ(GPⅥ)的研究进展

血小板膜糖蛋白Ⅵ（GPⅥ）是血小板表面重要的胶原受体，能介导血小板与胶原的初期黏附，产生信号转导，提高整合蛋白受体亲合力，引起血小板聚集和血栓形成。抑制GPⅥ功能可显著抑制胶原诱导的血小板黏附、聚集和血栓形成，故GPⅥ已成为研制新型抗血小板药物的主要靶点。过去几年中，对血小板和胶原相互作用的研究取得很大进展，本文就GPⅥ的结构，GPⅥ的功能，GPⅥ功能相关因素，GPⅥ与临床等作一综述。     

Presence of platelet-associated anti-glycoprotein (GP)VI autoantibodies and restoration of GPVI expression in patients with GPVI deficiency

Summary.  Background:  Glycoprotein (GP)VI deficiency is a rare platelet disorder with a mild bleeding tendency. However, its pathophysiology remains unclear. Objectives:  We characterized a novel GPVI-deficient patient with immune thrombocytopenic purpura and searched for the presence of anti-GPVI autoantibodies in this and another patient with GPVI deficiency. Methods and results:  A 12-year-old Japanese girl (case 1) with moderate thrombocytopenia and mild bleeding showed selectively impaired collagen-induced platelet aggregation. Flow cytometric analysis indicated that the patient had a defect in the expression of GPVI–FcRγ. An eluate of her platelet-associated IgG contained anti-α_IIbβ₃ autoantibodies. Moreover, using GPVI–FcRγ-transfected cells, we unexpectedly identified anti-GPVI antibodies against the soluble ectodomain of GPVI in the eluate, despite the patient's GPVI deficiency. In contrast, anti-GPVI antibodies were not detectable in her plasma. In another case of GPVI deficiency (case 2) without detectable plasma anti-GPVI antibodies, we again detected platelet-associated anti-GPVI antibodies. In a 2-year follow-up of case 1, the platelet count increased to within the normal range and the bleeding tendency improved. Interestingly, GPVI was again expressed on her platelets, in association with a decrease in the relative amount of anti-GPVI antibodies. Conclusions:  This is the first demonstration of platelet-associated anti-GPVI antibodies in GPVI-deficient subjects, in one case with spontaneous restoration of GPVI expression. These results strongly suggest an autoimmune mechanism in GPVI deficiency.     

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor gamma-chain complex

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.     

A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo

Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet-vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by approximately 89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet-collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.     

Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model

Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets. Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during thrombus formation in live mice. In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during thrombus formation following laser-induced injury in the cremaster muscle microcirculation. In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited. The kinetics of platelet activation and platelet accumulation were similar in FcRgamma(-/-) mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice. Our results indicate that the tissue factor-mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed. In the tissue factor-mediated pathway, vWF plays a role in platelet accumulation during thrombus formation but is not required for platelet activation in vivo.     

A synthetic peptide derived from the sequence of a type I collagen receptor inhibits type I collagen-mediated platelet aggregation.

A synthetic peptide-1, an 18 amino acid residue peptide derived from a hydrophilic domain of a cloned platelet type I collagen receptor, was used to study the role of the receptor on types I and III collagen-induced platelet aggregation and the release of ATP. The peptide inhibits the type I, but not the type III, collagen-induced platelet aggregation and the release of ATP in a dose-dependent manner. The [125I]peptide-1 specifically binds to type I collagen-coated microtiter wells in a dose-dependent manner (with Kd = 10 nM). The binding of [125I]peptide-1 can be inhibited by an excess of unlabeled peptide-1 suggesting that the binding is specific. The labeled peptide-1 does not bind to type III collagen-coated microtiter wells. Results from an enzyme-linked immunosorbent assay show that the peptide reacts with the poly- and monoclonal antibodies raised against the purified platelet type I collagen receptor (Mr 65 kD). The peptide also inhibits the adhesion of platelets on type I collagen matrix and rabbit aortic segments in a dose-dependent manner. These results suggest that the reactive site of the platelet receptor for type I collagen resides in this portion of the molecule.     

Adhesive surface determines raft composition in platelets adhered under flow

Adhesion to von Willebrand factor (VWF) induces platelet spreading, whereas adhesion to collagen induces aggregation. Here we report that cholesterol-rich domains (CRDs) or rafts play a critical role in clustering of receptors that control these responses. Platelets adhered to VWF and collagen show CRDs concentrated in filopodia which contain both the VWF receptor glycoprotein (GP) Ibalpha and the collagen receptor GPVI. Biochemical analysis of CRDs shows a threefold enrichment of GPIbalpha (but not GPVI) in VWF-adhered platelets and a fourfold enrichment of GPVI (but not GPIbalpha) in collagen-adhered platelets. Depletion of cholesterol (i) leaves the initial adhesion unchanged, (ii) inhibits spreading on VWF and aggregate formation on collagen, (iii) leaves filopodia formation intact, and (iv) reduces the localization in filopodia of GPIbalpha but not of GPVI. These data show that the adhesive substrate determines the composition of CRDs, and that cholesterol is crucial for redistribution of GPIbalpha but not of GPVI.     

Identification of a unique type of thrombopathy of human platelets: defect in the exposure of active fibrinogen receptors in a patient with Friedreich's ataxia

Platelets of a patient with Friedreich's ataxia have been investigated because of a codiagnosis of thrombasthenia. No aggregation occurred in response to adenosine diphosphate, platelet activating factor-acether, a stimulatory antiplatelet monoclonal antibody, or phorbol myristate acetate, although platelet aggregation could be induced with thrombin, the calcium ionophore A23187, or high concentrations of collagen. Shape change, adenosine triphosphate secretion, and the responses of the platelets' protein phosphorylation systems to all agonists were normal. Immunologic analysis of the patient's radiolabeled platelet surface proteins revealed normal levels of glycoproteins IIB and IIIa. However, no iodine 125-fibrinogen binding occurred after stimulation of the patient's platelets with adenosine diphosphate. In contrast, pretreatment of the patient's platelets with the proteolytic enzyme alpha-chymotrypsin resulted in the exposure of active 125I-fibrinogen binding sites. The patient's platelets exhibited normal aggregation to fibrinogen after their pretreatment with chymotrypsin and with elastases derived either from porcine pancreas or from human granulocytes. A murine monoclonal antibody directed against the human platelet membrane glycoproteins IIb and IIIa calcium-dependent epitope and rabbit polyclonal anti-human platelet membrane and human anti-P1A1 antibodies immunoprecipitated glycoproteins IIb and IIIa and a 66 kd cleavage product of glycoprotein IIIa from sodium dodecyl sulfate-Triton X-100 extracts of the patient's proteolytically treated platelets. The patient appears to exhibit a unique type of thrombopathy involving a defect in the exposure of fibrinogen receptors. The association between the neurologic disorder and the platelet defect is still unclear.     

Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs

Summary.  Background: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. Objectives: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc₂) but not to GPVI monomer (GPVIex) through a phage display method. Results: Ssix Fabs were found: B–F, only reactive with GPVI-Fc₂, and A, mainly reactive with GPVI-Fc₂, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc₂ binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc₂. Adding the anti-GPVI monoclonal antibody 204-11 increased the B _max of m-Fab-F binding to GPVI-Fc₂, suggesting that 204-11 binds to GPVI-Fc₂ molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. Conclusions: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.     

A new variant of Glanzmann''s thrombasthenia (Strasbourg I). Platelets with functionally defective glycoprotein IIb-IIIa complexes and a glycoprotein IIIa 214Arg----214Trp mutation.

We describe a new variant of Glanzmann's thrombasthenia (variant Strasbourg I). The patient (M.S.) showed an absence of platelet aggregation to ADP, thrombin, and collagen, and a decreased clot retraction. Platelet fibrinogen was approximately 20% of normal levels. ADP-stimulated platelets bound markedly reduced amounts of soluble fibrinogen and platelet adhesion to surface-bound fibrinogen was defective. Normal to subnormal amounts of glycoprotein (GP) IIb-IIIa (alpha IIb beta 3) complexes, the platelet fibrinogen receptor, were revealed by SDS-PAGE, crossed immunoelectrophoresis, and antibody binding. However, the complexes were unusually sensitive to dissociation with EDTA at room temperature. Furthermore, flow cytometry showed that the platelets failed to bind the activation-dependent monoclonal antibody, PAC-1, after stimulation. In contrast, an RGDS-containing peptide induced significant binding of the anti-ligand-induced binding site antibody, D3GP3, suggesting the presence of a functional RGD binding domain on the patient's GPIIb-IIIa complex. Sequence analysis was performed after polymerase chain reaction amplification of selected patient's GPIIIa exons, and of the patient's platelet GPIIb and GPIIIa mRNAs. A point mutation (C to T) was localized in exon D (iv) of GPIIIa that resulted in an 214Arg to 214Trp amino acid substitution. The defect has been inherited from the parents who are heterozygous for the same mutation. This substitution points to an essential amino acid in a region of GPIIIa involved in the binding of fibrinogen and influencing the Ca(2+)-dependent stability of the GPIIb-IIIa complex.     

Globular adiponectin induces platelet activation through the collagen receptor GPVI-Fc receptor γ chain complex

Summary.  Background:  The adipocyte-derived cytokine, adiponectin (Ad), exerts potent vascular effects, although the direct effects of Ad on blood platelets are unclear. Objective:  The influence of globular Ad (gAd) on blood platelet function was investigated. Research design and methods:  We measured platelet aggregation and tyrosine phosphorylation signaling events in human and mouse platelets. The ability of gAd to activate Glycoprotein VI (GPVI) activity was determined with a NFAT luciferase reporter assay. Results:  gAd, but not full length Ad, induced rapid aggregation and granule secretion of human and mouse platelets through a pathway that is ablated under conditions of Src kinase inhibition, indicating a tyrosine kinase-dependent mechanism. Consistent with this, gAd stimulates rapid tyrosine phosphorylation of several proteins in human and mouse platelets. The pattern of increase in tyrosine phosphorylation was similar to that induced by collagen, with the tyrosine kinase Syk and PLCγ2 being identified among the list of tyrosine phosphorylated proteins. As collagen activates platelet through the GPVI-Fc receptor γ-chain (FcRγ) complex, we used FcRγ null platelets (which also lack GPVI) to explore the mechanism by which gAd stimulates platelets. Stimulation of tyrosine phosphorylation and platelet aggregation by gAd was abolished in FcRγ null platelets and markedly reduced in the absence of PLCγ2. Further, GPVI was confirmed as a collagen receptor for gAd by increased luciferase activity in Jurkat T-cells transfected with GPVI. Conclusions:  We identify gAd as a novel ligand for GPVI that stimulates tyrosine kinase-dependent platelet aggregation. Our data raise the possibility that gAd may promote unwanted platelet activation at sites of vascular injury.     

Platelet cold agglutinins: a flow cytometric analysis.

Spontaneous EDTA-independent cold platelet agglutination is a rare phenomenon that produces pseudothrombocytopenia when blood samples are analyzed in automated cell counters. We report a case of platelet cold agglutinins and an analysis by flow cytometry. A 49 year old woman presented with abnormal vaginal bleed secondary to uterine fibroids. Platelet clumping was observed in blood samples taken in EDTA-, heparin- and citrate-containing tubes. In flow cytometric tests, patient serum agglutinated 16% of normal platelets at 22 degrees C, and 7% of platelets after incubation at 37 degrees C; in contrast, 3% and < 1% of platelets were agglutinated at 22 and 37 degrees C, respectively, after incubation with normal serum. Minimal agglutination (< 10%) was observed with patient serum at a titre of 1:5 or at temperatures > 30 degrees C. After incubation at 4 degrees C, IgM antibody and C3 were increased on the patient's platelets; no significant amount of IgM or C3 was detected on normal platelets. The specificity of the platelet cold agglutinin was determined by competitive inhibition by monoclonal anti-CD41(GPIIbIIIa). Before the addition of monoclonal antibody, patient's serum agglutinated 16% of normal platelets at 22 degrees C; after addition of anti-CD41 only 2% of the platelets were agglutinated. This blocking effect was not observed with anti-CD42. The patient's platelets functioned normally as determined by CD62 and CD63 expression in response to thrombin, normal platelet aggregation in response to collagen, ADP, and ristocetin, and a normal template bleeding time. In summary, platelet agglutination by a platelet cold agglutinin was quantitated by flow cytometry, the responsible antibody was characterized as a low titre IgM with minimal activity > 30 degrees C, and competitive binding studies supported the GPIIbIIIa complex as the binding site for the antibody. Since the antibody did not affect platelet function, we believe that these patients will not suffer complications from their platelet cold agglutinin, but it could pose a problem under circumstances such as cardiac surgery with hypothermia.     

Platelet glycoprotein VI facilitates experimental lung metastasis in syngenic mouse models

Summary.  Background : Glycoprotein (GP)VI is a key receptor for collagen on the platelet surface. It is a member of the immunoglobulin superfamily and is uniquely expressed on the surface of platelets, where it is assembled with the immunoreceptor tyrosine activation motif subunit, FcR-γ. We have previously reported the generation of a murine model of GPVI deficiency that revealed profound defects in collagen-induced platelet aggregation and in platelet activation following adhesion to collagen. Beyond the hemostasis/thrombosis paradigm, platelet receptors are emerging as significant participants in tumorigenesis and inflammation. Objective : In the current study, we have evaluated a role for platelet GPVI in primary tumor growth and experimental metastasis. Methods : Primary tumor induction and experimental metastasis assays were performed using syngenic immunocompetent animals and tumor cells derived from the C57BL/6J mouse strain in wild-type (C57BL/6J) and N10 C57BL/6J congenic GPVI-deficient mice. Results : Using either a Lewis lung carcinoma (D121) or melanoma (B16F10.1) cell line, we observed an approximately 50% reduction in the number of visible tumor foci in GPVI-deficient mice as compared with control C57BL/6J mice. Additional studies were performed to compare the size of subcutaneously implanted tumor cells, that is, primary tumor growth. Here, we observed no noticeable size difference when comparing the presence or absence of platelet GPVI. Conclusions : The results demonstrate that the presence of platelet GPVI facilitates experimental tumor metastasis but does not contribute to the growth of primary tumors.     

Establishment of a flow cytometric assay for determination of human platelet glycoprotein VI based on a mouse polyclonal antibody

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. Recent studies suggest that the surface density of GPVI is related to the activation of platelets by collagen. To measure the level of GPVI on platelets, a mouse polyclonal antibody BJ010 was prepared using an amplified fragment of extracellular domain in GPVI. The specific reactivity of BJ010 was identified by anti-GPVI specific monoclonal antibody 11a12 using immunoprecipitation, sandwich enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The antibody BJ010 recognized both native and denatured human GPVI so that it was used to set up a flow cytometric assay to detect the level of GPVI in normal subjects. The relative level of GPVI on platelets was detected in 101 healthydonors. The median geometric mean fluorescence intensity (GMFI) of platelet GPVI level was 57.7. The variation between minimum and maximum values of platelet GPVI and integrin alpha2beta1 were found to be 3.5- and 4.1-fold, respectively, in the normal subjects. There was a week correlation between the amount of GPVI and integrin alpha2beta1 on platelet surfaces. For the method, the intraassay and interassay coefficient of variation was 6.3% and 8.8%, respectively. The flow cytometric assay described here provides a simple, reliable, reproducible, and readily available means of quantitation of collagen receptor GPVI density on the platelet surface in a larger number of blood samples.