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.     

Heparin-induced thrombocytopenia

Summary.  Heparin-induced thrombocytopenia (HIT) is not only a common but also a potentially serious drug adverse effect. Unlike other drug-induced thrombocytopenias, HIT does not usually cause bleeding, but instead causes thrombosis. Thrombosis in HIT can lead to limb gangrene (requiring leg amputation) or even death. HIT is mediated by an antibody that recognizes an epitope on the platelet factor 4 (PF4)–heparin complex. The antibody–PF4–heparin complex binds to FcγRII receptors on the platelet surface and cross-links the receptors. This induces intense platelet activation and platelet aggregation, and simultaneously activates blood-coagulation pathways. These changes are probably the basis of the thrombotic events in HIT. Diagnosis of HIT should be made mainly on clinical criteria but should be confirmed whenever possible by laboratory tests, particularly in patients with comorbid conditions, in whom the diagnosis of HIT cannot be made with certainty without testing. The tests for HIT antibodies are either immunoassays (e.g. ELISA), or functional tests, (e.g. ¹⁴C-serotonin release assay). Once a clinical diagnosis of HIT is made, heparin should be ceased immediately and treatment with an alternative anticoagulant (such as danaparoid, r-hirudin or argatroban) commenced. This should continue for at least 5 days unless the diagnosis of HIT is subsequently proven to be incorrect. Warfarin should also be commenced when the patient is clinically stable and thrombosis is under control. There should be an overlap of a few days between warfarin and the alternative anticoagulant therapy.     

Platelet FcγRIIA HIS131ARG polymorphism and platelet function: antibodies to platelet-bound fibrinogen induce platelet activation

Summary.  The His131Arg polymorphism of platelet FcγRIIA affects the binding affinity of certain IgG subclasses. The Arg131 allele has been associated with (auto)immune thrombocytopenia and heparin-induced thrombocytopenia in some studies. Because FcγRIIA can transmit platelet activation signals, we studied platelet responsiveness from 73 healthy donors to determine if this polymorphism modulated platelet function. Platelet function was studied by agonist and shear-induced activation, and standard aggregation. FcγRIIA was genotyped by allele-specific PCR. Compared with His131, the Arg131 allele was associated with significantly greater binding of activation-dependent antibodies. This effect was most prominent for the receptor-induced binding site (RIBS) antibodies F26 ( P  < 0.0001) and RIBS1 ( P  = 0.0057), and the ligand-induced binding site antibody LIBS1 ( P  = 0.0367). Unexpectedly, Arg131-positive platelets did not show greater fibrinogen binding, platelet aggregation or shear-induced platelet activation. We considered whether enhanced Fc binding and FcγRIIA cross-linking were responsible for those discrepancies. The increased binding of the two RIBS antibodies to the Arg131 isoform was abolished by blocking FcγRIIA, and the FcγRIIA genotype effect on F26 IgG binding was lost when F26 F(ab')₂ fragments were used. Furthermore, intact F26 and RIBS1 IgG directly and specifically induced P-selectin expression, and this effect was greatest in Arg131-positive platelets. We concluded that (a) the His131Arg polymorphism of FcγRIIA does not affect intrinsic platelet reactivity; (b) RIBS antibodies are able to cross-link FcγRIIA and activate platelets, and this activation has a modest effect on Arg131 platelets; and (c) flow cytometric based platelet assays may need to compensate for this FcγRIIA His131Arg effect on platelet activation.     

Early atherosclerosis and IgG2 to bacteria are associated with FcγRIIa genotype in non-smokers

Background  Involvement of low density lipoprotein (LDL) immune complexes (ICs) in atherogenesis has been proposed. Human FcγRIIa receptor (CD32) plays a crucial role in the phagocytosis of IgG₂ ICs and a functional point mutation 131His/Arg diminishes IgG₂ binding to the receptor.
Study design  We examined FcγRIIa-131His/Arg polymorphism, IgG₂ antibody titres to oxidized low-density lipoprotein (OxLDL) and Streptococcus pneumoniae cell wall polysaccharide (CWPS) and subclinical atherosclerosis in a large cohort of Finnish subjects ( n  = 1041).
Results  Non-smoking subjects with homozygous 131His/His genotype had more premature atherosclerosis ( P  =   0·004) and higher IgG₂ to bacterial CWPS ( P  =   0·002) compared with other genotypes. Smoking subjects had significantly higher intima-media thickness (IMT) than that of non-smokers ( P  <   0·001) and genotype-dependent associations were indistinct. There was no association between FcγRIIa genotype and antibody titres to OxLDL.
Conclusions  Our data demonstrate that FcγRIIa 131His/Arg polymorphism is associated with subclinical atherosclerosis in non-smoking subjects. Furthermore, FcγRIIa genotype is associated with IgG₂ titres to bacterial CWPS, but not to OxLDL. These data propose possible involvement of FcγRIIa receptor in atherogenesis.     

Elucidating the role of Staphylococcus epidermidis serine–aspartate repeat protein G in platelet activation

Summary.  Background: Staphylococcus epidermidis is a commensal of the human skin that has been implicated in infective endocarditis and infections involving implanted medical devices. S. epidermidis induces platelet aggregation by an unknown mechanism. The fibrinogen-binding protein serine–aspartate repeat protein G (SdrG) is present in 67–91% of clinical strains. Objectives:  To determine whether SdrG plays a role in platelet activation, and if so to investigate the role of fibrinogen in this mechanism. Methods : SdrG was expressed in a surrogate host, Lactococcus lactis , in order to investigate its role in the absence of other staphylococcal components. Platelet adhesion and platelet aggregation assays were employed. Results:    L. lactis expressing SdrG stimulated platelet aggregation (lag time: 2.9 ± 0.5 min), whereas the L. lactis control did not. L. lactis SdrG-induced aggregation was inhibited by α_IIbβ₃ antagonists and aspirin. Aggregation was dependent on both fibrinogen and IgG, and the platelet IgG receptor FcγRIIa. Preincubation of the bacteria with Bβ-chain fibrinopeptide inhibited aggregation (delaying the lag time six-fold), suggesting that fibrinogen acts as a bridging molecule. Platelets adhered to L. lactis SdrG in the absence of fibrinogen. Adhesion was inhibited by α_IIbβ₃ antagonists, suggesting that this direct interaction involves α_IIbβ₃. Investigation using purified fragments of SdrG revealed a direct interaction with the B-domains. Adhesion to the A-domain involved both a fibrinogen and an IgG bridge. Conclusion:  SdrG alone is sufficient to support platelet adhesion and aggregation through both direct and indirect mechanisms.     

Highly accumulated platelet vascular endothelial growth factor in coagulant thrombotic region

Summary.  Background : Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm. Objectives : To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis. Methods : The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed. Results and conclusions : Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-α, and TGF-β.     

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.     

Prothrombotic factors enhance heparin-induced thrombocytopenia and thrombosis in vivo in a mouse model

BACKGROUND: Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common cause of life- and limb-threatening thrombosis. The development of antibodies that react with complexes of heparin and platelet factor 4 (PF4) is fundamental to the development of the disease. However, anti-PF4/heparin antibodies are far more common than is HIT/T and there is less understanding of the factors that contribute to thrombosis in only a subset of patients. OBJECTIVES: Both qualitative and quantitative differences in multiple factors (e.g. antibodies, heparin and platelets) may influence the clinical course of patients who develop anti-PF4/heparin antibodies. We examined the hypothesis that host-specific factors, such as comorbid prothrombotic conditions, would exacerbate the pathologic effects of anti-PF4/heparin antibodies. METHODS AND RESULTS: A mouse model transgenic for human Fcgamma RIIa and PF4 and null for mouse PF4 was used to study the influence of prothrombotic conditions on the effects of anti-PF4/heparin antibodies in vivo. To simulate a prothrombotic milieu, mice were fed a hypercholesterolemic diet (HD). HD-fed mice had elevated plasma cholesterol, increased platelet reactivity and increased endothelial activation relative to mice fed a standard diet (SD). Age- and sex-matched mice from each diet group were treated with an anti-PF4/heparin antibody and heparin. HD-fed mice developed more severe thrombocytopenia than similarly treated SD-fed mice. Mice with moderate to severe thrombocytopenia had elevated plasma levels of thrombin-antithrombin complexes, indicative of increased thrombin generation in vivo. Platelet-fibrin thrombi were observed in multiple organs of HD-fed mice that developed severe thrombocytopenia. CONCLUSIONS: Host-specific factors, such as prothrombotic changes in platelet reactivity and/or endothelial activation, may influence the development of thrombosis in a subset of patients who develop anti-PF4/heparin antibodies.     

Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice

Summary.  In order to investigate the role of the platelet P2Y₁ receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [³³P]2MeSADP in the presence or absence of the selective P2Y₁ antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y₁ receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y₁ receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y₁ receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y₁ expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y₁ receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y₁ receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.     

Intravenous immunoglobulin as an adjunct therapy in persisting heparin-induced thrombocytopenia

Heparin induced thrombocytopenia (HIT) is a serious adverse drug reaction caused by transient antibodies against platelet factor 4 (PF4)/heparin complexes, resulting in platelet activation and potentially fatal arterial and/or venous thrombosis. Most cases of HIT respond to cessation of heparin and administration of an alternative non-heparin anticoagulant, but there are cases of persisting HIT, defined as thrombocytopenia due to platelet activation/consumption for greater than seven days despite standard therapy. These patients remain at high risk for thrombotic events, which may result in limb-loss and mortality. Intravenous immunoglobulin (IVIg) has been proposed as an adjunct therapy for these refractory cases based on its ability to saturate FcγRIIa receptors on platelets, thus preventing HIT antibody binding and platelet activation. We describe 2 cases of persisting HIT (strongly positive antigen and functional assays, and persisting thrombocytopenia >7 days) with rapid clinical response to IVIg. We performed in-vitro experiments to support IVIg response. Healthy donor platelets (1?×?10e6) were treated with PF4 (3.75?μg/mL) for 20?min followed by 1-hour incubation with patients’ sera. Platelet activation with and without addition of IVIg (levels equivalent to those reached in a patient after treatment with 2?gm/Kg) was evaluated in the PF4-dependent P-selectin expression assay (PEA). A significantly decreased platelet activation was demonstrated after the addition of IVIg to both patient samples, which correlated well with the rapid clinical response that each patient experienced. Thus, our study supports the use of IVIg as an adjunct therapy for persisting HIT.     

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.     

Role of FcγRIIa (CD32) in IgG anti-RhD-mediated red cell phagocytosis in vitro

Summary. To test the role of FcγRIIa in IgG anti-RhD-mediated phagocytosis three IgGl and two IgG3 human monoclonal anti-D antibodies were tested for ability to mediate binding/phagocytosis of cDE/cde and -D-/-D- red cells by FcγRIIa-R131 and FcγRIIa-H131 cDNA-transfected 3T6 fibroblasts. Both IgG3 monoclonal antibodies brought about -D-/-D- cell interaction with IIa-transfected fibroblasts, while only one of them, Og3, mediated binding of cDE/cde targets. Although FcγRIIa expression was three times greater on IIa-R131 than on IIa-H131 fibroblasts, the latter bound significantly more Og3-coated cDE/cde- and IgG3 anti-D-sensitized -D-/-D- cells, respectively, than the former effectors and showed some phagocytosis of the -D-/-D-targets. IgGl anti-D antibodies were inactive in mediating red cell interaction with the fibroblasts. Moreover, monoclonal anti-FcγRII IV.3 partially inhibited the phagocytosis by adult or fetal monocytes of Og3-sensitized cDE/cde cells. FcγRIIa-H/H131 monocytes exhibited higher phagocytic indices towards these targets than monocytes of other IIa allotypes. The results indicate that FcγRIIa can participate in the phagocytosis of red cells coated with IgG3 anti-D; in this case the allotype of the receptor will modify the extent of red cell destruction.     

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.     

False-positive tests for heparin-induced thrombocytopenia in patients with antiphospholipid syndrome and systemic lupus erythematosus

Summary.  Background : Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy that can be associated with arterial or venous thrombosis and is caused by antibodies against platelet factor 4 (PF4)–heparin complex. Patients with antiphospholipid syndrome (APS) have been reported with positive tests for PF4–heparin complex antibodies by antigen assay. Whether such patients can be treated with heparin is a dilemma. Objectives : To determine the incidence and nature of the HIT immune reaction in patients with APS and/or systemic lupus erythematosus (SLE). Methods : Antibodies against PF4–heparin complex were assayed by particle gel immunoassay (PaGIA), or enzyme immunoassay (EIA) with or without an excess of heparin. EIA for PF4 alone was also performed. Functional assays for HIT, that is, heparin-induced platelet activation (HIPA) and heparin-induced platelet aggregation, were also performed. Results : In 32 of 42 patients (76.2%) with APS, APS and SLE, SLE, or SLE with antiphospholipid antibodies, EIA IgG or PaGIA for PF4–heparin complex antibodies were positive. Of these 32 samples, 26 (81.3%) tested positive for anti-PF4 antibodies. All 24 samples that were positive for PF4–heparin complex by EIA IgG were also positive for EIA IgG in the presence of heparin excess, and all were negative by the HIPA and heparin-induced platelet aggregation tests. Conclusion : A large proportion of patients with APS and/or SLE give false-positive HIT antigen test results that are presumably related to autoantibodies against PF4, which can be distinguished from true HIT antibodies by EIA for PF4–heparin complexes tested with heparin excess, and by functional assays.     

Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure

Summary.  Background:  Platelet membrane phosphatidylserine (PS) is considered to be essential for hemostasis and thrombosis, but the in vivo topography of platelet PS has not been characterized. We hypothesized that platelet PS exposure would be identified on adherent platelets at the site of vascular injury and that blockade of PS would impede hemostasis and thrombosis. Objective:  To localize and estimate the extent of platelet PS exposure and evaluate the impact of PS blockade in vivo . Methods:  Lactadherin, a PS-binding milk protein, was utilized together with annexin  V to detect both partial and complete membrane PS exposure on platelets in a mouse model of thrombosis and to evaluate the functional need for PS. Preliminary experiments were performed with synthetic membranes and with purified platelets. Results:  The number of lactadherin-binding sites on synthetic membranes was proportional to PS content, whereas annexin  V required a threshold of 2.5–8% PS. Approximately 95% of thrombin-stimulated platelets exposed PS, but the quantity was below the threshold for annexin  V binding at physiologic Ca²⁺ concentrations. In mice, most adherent and aggregated platelets on the walls of ferric chloride-treated mesenteric veins exposed low levels of PS, rather than having complete exposure. In mice, blockade of PS with lactadherin inhibited platelet prothrombinase and factor Xase activity, and prolonged tail bleeding time and the time to carotid artery thrombosis. Conclusions:   In vivo PS exposure contributes to both hemostasis and thrombosis. In this model of vascular injury, most platelets exhibit partial rather than complete PS exposure.     

Platelet receptor recognition and cross-talk in collagen-induced activation of platelets

Summary.  Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin α₂β₁ and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR γ-chain and/or FcγRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and α₂β₁ at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.     

Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis

Beyond their prominent role in hemostasis and thrombosis, platelets are increasingly recognized as having immunologic functions. Supporting this, human platelets express FcγRIIA (CD32a), a low‐affinity Fc receptor (FcR) for the constant region of IgG that recognizes immune complexes (ICs) and IgG‐opsonized cells with high avidity. In leukocytes, FcγRIIA engagement initiates strong effector functions that are key for immune and inflammatory responses, including cytokine release, antibody‐dependent cell‐mediated killing of pathogens, and internalization of ICs. However, the physiologic relevance of platelet‐expressed FcγRIIA has received little attention in previous reviews on FcRs. This article summarizes and discusses the available information on human platelet FcγRIIA. The importance of this receptor in heparin‐induced thrombocytopenia, a prothrombotic adverse drug effect, is well documented. However, studies demonstrating platelet activation by IgG‐opsonized bacteria point to the physiologic relevance of platelet FcγRIIA in immunity. In this context, platelet activation and secretion may facilitate both a direct antimicrobial function of platelets and crosstalk with other immune cells. Additionally, a role for platelet FcγRIIA in IgG‐independent hemostasis and physiologic thrombosis, by means of amplifying integrin α_IIbβ₃ outside‐in signaling, has also been proposed. Nonetheless, the thrombotic complications found in some infective and autoimmune diseases may result from unbalanced FcγRIIA‐mediated platelet aggregation. Moreover, FcγRIIA is not expressed in mice, and thrombocytopenia and/or thrombotic events found after drug administration can only be recapitulated by the use of human FcγRIIA‐transgenic mice. Altogether, the available data support a functional role for platelet FcγRIIA in health and disease, and emphasize the need for further investigation of this receptor.     

Heparin but not citrate anticoagulation of blood preserves platelet function for prolonged periods

Summary.  Background:  Current guidelines state that platelet aggregation studies should be conducted within 4 h of venepuncture because of the decline in sensitivity to platelet agonists. This constrains studies of platelet activity in clinical situations where samples need to be transported or there are unavoidable delays prior to assessment. Objectives:  The aim of the present study was to compare systematically the responses of platelets stored in the presence of either citrate or heparin, the two most widely used anti-coagulants, using a range of standard techniques. Methods:  Blood was taken from healthy volunteers and either assessed immediately or stored at ambient temperature (18–25 °C) for 24 h. Platelet reactivity to a range of agonists was determined by a combination of 96-well plate techniques; light transmission aggregometry, thrombi adhesion, ATP and ADP release, and TxA₂ release; by whole blood aggregometry; and by PFA-100. Results and conclusions:  Testing using 96-well plate techniques allowed for the simultaneous measurement of responses to multiple concentrations of multiple agonists. The responses of platelets from blood anti-coagulated with heparin were predominantly preserved in all assays after 24 h storage, whereas, responses of platelets stored in blood anti-coagulated with citrate were greatly diminished. Consequently, anti-coagulation with heparin, but not citrate, preserves platelet responses for up to 24 h as determined by a range of techniques.     

Abnormal platelet function in C3-deficient mice

Summary.  Background and Objectives:  The complement system is a biochemical cascade composed of several plasma proteins that can interact with endothelial cells and blood cells, including platelets. In order to investigate the effect of the complement system on platelets, we studied platelet function in C3-deficient mice that lack complement activity. Method and Results:  Tail-cut bleeding time was prolonged and platelet aggregation in response to protease-activated receptor-4 (PAR4) peptide was decreased in C3-deficient mice as compared with wild-type littermates. Platelet aggregation in response to other agonists (ADP and collagen) was similar between C3-deficient mice and their normal littermates. Isolated platelets from wild-type mice aggregate less in C3-deficient plasma than in normal plasma, and, conversely, addition of plasma from wild-type mice or plasma-purified C3 improved aggregation of C3-deficient platelets. We also monitored the formation of murine arteriole or venule thrombi in an intravital microscopy thrombosis model. We found that C3-deficient mice had a significantly delayed thrombotic response in arterioles as compared with their wild-type littermates. Furthermore, thrombi in C3-deficient mice were less stable and embolized more frequently than those in wild-type mice. Conclusions:  Platelets of C3-deficient mice have subnormal function, resulting in a prolonged tail-cut bleeding time and delayed thrombosis after vessel wall injury.     

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.