Stephanie M. Jung

Platelets Interact with Soluble and Insoluble Collagens through Characteristically Different Reactions

Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin α2β1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin α2β1 antibody). Each platelet has 1500–3500 soluble collagen binding sites, with a dissociation constant of 3.5–9 × 10−8 m. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin α2β1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin α2β1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.

Activation of the Platelet Collagen Receptor Integrin α2β1: Its Mechanism and Participation in the Physiological Functions of Platelets

When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.

Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood

Background: Platelet collagen receptor GPVI likely functions as a dimer rather than a monomer. Results: Preformed GPVI dimers, but not monomers, in resting platelets bind specific collagen sequences and are essential for platelet adhesion and activation. Conclusion: Constitutive GPVI dimers on resting platelets support platelet adhesion to collagen and activation. Significance: Resting platelets bind collagen through GPVI dimers, allowing immediate initiation of thrombus formation. The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (Kd) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)10-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ∼29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ∼39 and ∼44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

Cloning and Expression of the Platelet-Specific Collagen Receptor Glycoprotein VI

Platelet glycoprotein VI (GP VI) was purified from platelet membranes and its internal amino acid sequences were determined. The cloned cDNA of GP VI indicates an open reading frame coding for 20 amino acid signal sequences and a mature protein of 319 amino acids. Its extracellular region has two Ig-like domains and a mucin-like, Ser/Thr-rich region, suggesting that GP VI is a member of the paired Ig-like receptor family. GP VI-transfected cells contained convulxin-(reactive) and antibody against recombinant GP VI-reactive protein bands that migrated at the same position as platelet GP VI in SDS/PAGE-electroblotting. These data indicate that the protein deduced from the cloned cDNA corresponds to platelet GP VI.

INTEGRIN-MEDIATED PLATELET ADHESION

Adhesion of platelets to the damaged subendothelium is a prerequisite reaction for the initiation of hemostasis in vivo. Platelet membranes contain high concentrations of integrins and other glycoproteins (GPs) that are involved in the platelet adhesion to the extracellular matrix. In the present review, we focus on two platelet integrins, integrin alphaIIb beta3 (GPIIb/IIIa) and integrin alpha2 beta1 (GPIa/IIa) because these integrins are major components of the platelet membrane proteins and are known to contribute to platelet adhesion to fibrin(ogen) and collagen surfaces, respectively. These integrins bind soluble ligands (fibrinogen or collagen) after platelets are activated but only have low affinity towards these ligands when platelets are in the resting state. We describe the binding properties of these integrins and discuss the mechanism for the activation of these integrins. Platelets can adhere to fibrin(ogen) or collagen immobilized on a surface. When platelets adhere to a collagen- or fibrin-coated surface, they become activated and form aggregates; this is especially prominent under flow conditions. We discuss the contribution of these integrins and non-integrin proteins, GPIb and GPVI, to the platelet adhesion on to the collagen surface, especially under flow conditions, a system that most closely approximates platelet adhesion in vivo.

Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators.

Flierl et al. show that phosphorothioate (PS) oligonucleotides activate platelets via interacting with the collagen receptor GPVI. As PS backbone modification is currently used for nucleotide-based drug candidates, the findings suggest that this widely used method may present a risk to patients in the form of arterial thrombosis.

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 Bmax 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.

Platelet Glycoprotein VI

Glycoprotein VI (GPVI) is a membrane glycoprotein unique to platelets and has been identified as a physiological receptor for collagen. Damage to a vessel wall exposes the subendothelial component collagen to platelets in the blood flow. Interaction of platelets with collagen via the receptor GPVI results in platelet activation and adhesion--the processes that are essential for thrombus formation. On the platelet surface, GPVI is present as a complex with the homodimeric Fc receptor y-chain (FcRgamma with a possible stoichiometry of two GPVI molecules and one FcRgamma dimer). When collagen binds to GPVI, a platelet activation cascade is initiated by tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif of FcRgamma and this phosphorylation induces the formation of a large complex composed from many signal-transducing proteins. In flow adhesion experiments that closely approximate physiological conditions, GPVI is essential for the formation of large platelet aggregates on collagen. However, GPVI-deficient patients or mice do not show any severe bleeding tendency. This suggests that a GPVI inhibitor would be able to inhibit thrombus formation but still not cause a significant bleeding tendency. Such an inhibitor would show promise as an anti-thrombotic agent for clinical use.

Platelet adhesion to collagen-coated wells: Analysis of this complex process and a comparison with the adhesion to matrigel-coated wells

The mechanisms of platelet adhesion to collagen type III-coated wells and Matrigel-coated wells were analyzed. The adhesion of 51Cr-labeled platelets to collagen-coated wells showed a biphasic pattern. The early stage of adhesion was inhibited by antibodies against platelet glycoprotein(GP)s Ia/IIa and VI. The later stage of platelet adhesion was inhibited by an antibody against the GPIIb/IIIa complex and a concomitant release of 14C-labeled serotonin was observed. The percentage of adhered platelets was increased when a higher platelet concentration was added in the reaction medium. These results indicated that the adhesion assay of platelets to collagen-coated wells was composed of two reactions: the first one is the platelet-collagen interaction that depends on GPIa/IIa and GPVI on the platelet surface; and the second reaction is the platelet-platelet interaction, platelet aggregation, which depends on GPIIb/IIIa. The adhesion of platelets to Matrigel-coated wells was indicated to involve platelet-Matrigel interactions that were partly dependent on the laminin in the Matrigel solution.

Characterization of a patient with glycoprotein (GP) VI deficiency possessing neither anti-GPVI autoantibody nor genetic aberration.

Summary.  Background: There have been only seven reported cases of glycoprotein (GP) VI deficiency. However, the pathogenesis of this disorder has not been well‐elucidated. Objectives: We characterized a novel patient with GPVI deficiency and used these platelets to investigate the role of GPVI in normal hemostasis. Patient: A 31‐year‐old female with immune thrombocytopenic purpura who had been suffering from mild bleeding diathesis even after recovery from thrombocytopenia. Results and conclusion: The patients platelets did not aggregate in response to either convulxin or collagen‐related peptide. Immunoblotting revealed complete absence of the GPVI molecule, whereas a significantly reduced but substantial amount of Fc receptor (FcR) γ‐chain was expressed. Platelet stimulation with convulxin did not induce tyrosine‐phosphorylation of FcR γ‐chain, indicating a defect in GPVI‐mediated signaling. Concerning the underlying pathogenesis, we found normal level of GPVI‐mRNA expression, no aberration of the sequence of the entire coding region of GPVI, and presence of degraded GPVI in her plasma. However, no anti‐GPVI autoantibody was detected either by the binding assay to GPVI‐Fc2 fusion protein or by immunoblotting/immunoprecipitation using the patients immunoglobulin. We thus consider that either a short‐time exposure to anti‐GPVI autoantibody or a continuous exposure to low titers of the autoantibody has resulted in persistent GPVI deficiency. Under high shear flow, the patients platelets could not form large aggregates, although initial platelet attachment was obviously observed. These results suggest that GPVI deficiency in this patient resulted in defective platelet thrombi development, manifesting as bleeding diathesis. Furthermore, our observations indicate that coordination of GPVI with integrin α2β1 is essential for physiological platelet thrombus formation.