Collagen-platelet interactions: recognition and signalling

The collagen-platelet interaction is central to haemostasis and may be a critical determinant of arterial thrombosis, where subendothelium is exposed after rupture of atherosclerotic plaque. Recent research has capitalized on the cloning of an important signalling receptor for collagen, glycoprotein VI, which is expressed only on platelets, and on the use of collagen-mimetic peptides as specific tools for both glycoprotein VI and integrin alpha 2 beta 1. We have identified sequences, GPO and GFOGER (where O denotes hydroxyproline), within collagen that are recognized by the collagen receptors glycoprotein VI and integrin alpha 2 beta 1 respectively, allowing their signalling properties and specific functional roles to be examined. Triple-helical peptides containing these sequences were used to show the signalling potential of integrin alpha 2 beta 1, and to confirm its important contribution to platelet adhesion. Glycoprotein VI appears to operate functionally on the platelet surface as a dimer, which recognizes GPO motifs that are separated by four triplets of collagen sequence. These advances will allow the relationship between the structure of collagen and its haemostatic activity to be established.     

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

The alpha 2 beta 1 integrin cell surface collagen receptor binds to the alpha 1 (I)-CB3 peptide of collagen

We have previously shown that platelets adhere to collagen substrates via a Mg2(+)-dependent mechanism mediated by the surface glycoprotein Ia-IIa (human leukocyte very late activation protein 2, alpha 2 beta 1 integrin) complex. The adhesion is specific for collagen and is supported by collagen types I, II, III, IV, and VI. Several other members of the integrin family of adhesive protein receptors recognize discrete linear amino acid sequences within their adhesive glycoprotein ligands. Experiments with both intact platelets and with liposomes containing the purified receptor complex indicated that the alpha 2 beta 1 receptor recognized denatured type I collagen in a Mg2(+)-dependent manner. To further localize the binding site, the alpha 1 and alpha 2 chains of type I collagen were purified by gel filtration and ion exchange chromatography and tested as adhesive substrates. Both the alpha 1(I) and alpha 2(I) chains effectively supported Mg2(+)-dependent platelet adhesion. The purified alpha 1(I) collagen chain was then subjected to cleavage with cyanogen bromide, and the resultant peptides were separated by chromatography on carboxymethylcellulose. Only the alpha 1(I)-CB3 fragment supported Mg2(+)-dependent platelet adhesion. The monoclonal antibody P1H5 which recognizes an epitope on the alpha 2 subunit of the integrin receptor and which inhibits the adhesion of both intact platelets and liposomes bearing the purified receptor to collagen also inhibited platelet adhesion to the alpha 1(I)-CB3 fragment. These results indicate that the alpha 2 beta 1 receptor recognizes a sequence of amino acids present in the alpha 1(I)-CB3 fragment of type I collagen. An identical or similar sequence likely mediates binding of the receptor to other collagen polypeptides.     

Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von Willebrand factor

Blood-sucking arthropods have evolved a number of inhibitors of platelet aggregation and blood coagulation. In this study we have molecularly and functionally characterized aegyptin, a member of the family of 30-kDa salivary allergens from Aedes aegypti, whose function remained elusive thus far. Aegyptin displays a unique sequence characterized by glycine, glutamic acid, and aspartic acid repeats and was shown to specifically block collagen-induced human platelet aggregation and granule secretion. Plasmon resonance experiments demonstrate that aegyptin binds to collagen types I-V (K(d) approximately 1 nm) but does not interact with vitronectin, fibronectin, laminin, fibrinogen, and von Willebrand factor (vWf). In addition, aegyptin attenuates platelet adhesion to soluble or fibrillar collagen. Furthermore, aegyptin inhibits vWf interaction with collagen type III under static conditions and completely blocks platelet adhesion to collagen under flow conditions at high shear rates. Notably, aegyptin prevents collagen but not convulxin binding to recombinant glycoprotein VI. These findings suggest that aegyptin recognizes specific binding sites for glycoprotein VI, integrin alpha2beta1, and vWf, thereby preventing collagen interaction with its three major ligands. Aegyptin is a novel tool to study collagen-platelet interaction and a prototype for development of molecules with antithrombotic properties.     

Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.     

Site-specific Phosphorylation of Kindlin-3 Protein Regulates Its Capacity to Control Cellular Responses Mediated by Integrin αIIbβ3

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin α_IIbβ₃, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), α_IIbβ₃ became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β₃ integrin and kindlin-3 in focal adhesions, and enhanced β₃ integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr⁴⁸² or Ser⁴⁸⁴ was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476–485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β₃ activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.     

Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP VI

alpha2beta1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-alpha2beta1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-alpha2beta1 integrin antibody. The combination of anti-alpha2beta1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-alpha2beta1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by alpha2beta1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both alpha2beta1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.     

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.     

A novel disintegrin-like domain of a high molecular weight metalloprotease inhibits platelet aggregation

Disintegrin is one of the functionally distinct domains in high molecular weight metalloproteases from various snake venoms and generally has an Arg-Gly-Asp (RGD) sequence that is recognized by specific cell surface integrins. A cDNA encoding the disintegrin-like domain of a snake venom metalloprotease was cloned, expressed in Pichia pastoris, and molecular function of the recombinant protein was characterized. The cDNA sequence indicated that the disintegrin-like domain contains an Asp-Glu-Cys-Asp (DECD) sequence in place of the RGD motif. The expressed disintegrin-like protein was designated as halydin and it was able to inhibit human platelet aggregation in a dose-dependent manner. Unlike other typical RGD-disintegrins, the recombinant non-RGD disintegrin, halydin, inhibited platelet aggregation by suppressing platelet adhesion to collagen rather than by blocking fibrinogen binding to glycoprotein (GP) IIb-IIIa on the platelet surface. Experimental evidence suggests that halydin binds to integrin alpha2beta1 on the platelet surface.     

A novel anticoagulant purified from fish protein hydrolysate inhibits factor XIIa and platelet aggregation

A novel fish protein having anticoagulant and antiplatelet properties was enzymatically extracted from the marine fish, yellowfin sole (Limanda aspera) and purified to homogeneity producing an overall purification fold of 206.6. MALDI-TOF mass spectroscopic and SDS-PAGE analysis identified the purified protein as 12.01 kDa single-chain monomeric protein. It inhibited the activated coagulation factor XII (FXIIa) by forming an inactive complex regardless of Zn2+ mediation, and was named, yellowfin sole anticoagulant protein (YAP). In addition, YAP act to antagonize platelet membrane glycoprotein integrin, to arrest platelet aggregation. However, YAP was not able to block the adhesion of platelets to collagen, which mediate via major collagen receptors, GPIa/IIa on platelet membrane. Furthermore, YAP did not possess plasminogen activator-like activity to activate fibrinolysis. In fact, our findings indicate that YAP binds with FXIIa and platelet membrane integrins to inhibit thrombosis in vitro.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption

The blood cell-specific kindlin-3 protein is required to activate leukocyte and platelet integrins. In line with this function, mutations in the KINDLIN-3 gene in man cause immunodeficiency and severe bleeding. Some patients also suffer from osteopetrosis, but the underlying mechanism leading to abnormal bone turnover is unknown. Here we show that kindlin-3-deficient mice develop severe osteopetrosis because of profound adhesion and spreading defects in bone-resorbing osteoclasts. Mechanistically, loss of kindlin-3 impairs the activation of β1, β2, and β3 integrin classes expressed on osteoclasts, which in turn abrogates the formation of podosomes and sealing zones required for bone resorption. In agreement with these findings, genetic ablation of all integrin classes abolishes the development of podosomes, mimicking kindlin-3 deficiency. Although loss of single integrin classes gives rise to podosomes, their resorptive activity is impaired. These findings show that osteoclasts require their entire integrin repertoire to be regulated by kindlin-3 to orchestrate bone homeostasis.     

G蛋白与整合素αⅡbβ3的双向信号转导

整合素是一类细胞表面受体家族分子,通过双向信号转导参与细胞与细胞外基质、细胞与细胞的粘附以及细胞的迁移.整合素αⅡbβ3(GPⅡb-Ⅲa)特异表达于巨核/血小板系,并且是其含量最多的膜糖蛋白,介导血小板的粘附、伸展、聚集等.G蛋白在整合素αⅡbβ3双向信号转导中发挥重要作用,其中较受关注的是:异源三聚体G蛋白和小G蛋白Rap1参与整合素αⅡbβ3的内向外信号转导;小G蛋白(Rho A、Rac等)和Gα13参与整合素αⅡbβ3的外向内信号转导.在蛋白质结构与功能关系的层面,本文总结了G蛋白的结构、分类、功能以及近年来G蛋白在整合素αⅡbβ3双向信号转导中作用的研究进展.     

A novel leukocyte adhesion deficiency III variant: kindlin-3 deficiency results in integrin- and nonintegrin-related defects in different steps of leukocyte adhesion

Leukocyte adhesion deficiency type III is a recently described condition involving a Glanzmann-type bleeding syndrome and leukocyte adhesion deficiency. This was ascribed to a defect of the FERMT3 gene resulting in abnormal expression of kindlin-3, a protein expressed in hematopoietic cells with a major role in the regulation of integrin activation. In this article, we describe a patient with a new mutation of FERMT3 and lack of kindlin-3 expression in platelets and leukocytes. We assayed quantitatively the first steps of kindlin-3-defective leukocyte adhesion, namely, initial bond formation, bond strengthening, and early spreading. Initial bond formation was readily stimulated with neutrophils stimulated by fMLF, and neutrophils and lymphocytes stimulated by a phorbol ester or Mn(2+). In contrast, attachment strengthening was defective in the patient's lymphocytes treated with PMA or Mn(2+), or fMLF-stimulated neutrophils. However, attachment strengthening was normal in patient's neutrophils treated with phorbol ester or Mn(2+). In addition, the patient's T lymphocytes displayed defective integrin-mediated spreading and a moderate but significant decrease of spreading on anti-CD3-coated surfaces. Patient's neutrophils displayed a drastic alteration of integrin-mediated spreading after fMLF or PMA stimulation, whereas signaling-independent Mn(2+) allowed significant spreading. In conclusion, the consequences of kindlin-3 deficiency on β(2) integrin function depend on both cell type and the stimulus used for integrin activation. Our results suggest looking for a possible kindlin-3 involvement in membrane dynamical event independent of integrin-mediated adhesion.     

Red cell ICAM-4 is a novel ligand for platelet-activated alpha IIbbeta 3 integrin

ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.     

Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets

Background

Brazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties.

Methods

In this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study.

Results

We demonstrated that relatively low concentrations of brazilin (1 to 10 μM) potentiated platelet aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 μM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin α_IIbβ₃ in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α₂β₁, respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules.

Conclusion

To the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.     

Integrin-independent tyrosine phosphorylation of p125(fak) in human platelets stimulated by collagen

Collagen fibers or a glycoprotein VI-specific collagen-related peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125(fak) (FAK), in human platelets. An integrin alpha(2)beta(1)-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O = Hyp) was without effect. Antibodies to the alpha(2) and beta(1) integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin alpha(IIb)beta(3). The intracellular Ca(2+) chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin alpha(2)beta(1) is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor alpha(IIb)beta(3) is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca(2+) and protein kinase C activity are essential intermediaries of FAK activation.     

Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen

Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α₂β₁, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen.     

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.