Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.     

The leech product saratin is a potent inhibitor of platelet integrin alpha2beta1 and von Willebrand factor binding to collagen

Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor, which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors glycoprotein VI and alpha2beta1 to collagen is critical for platelet activation and stable adhesion. Leeches, have evolved a number of inhibitors directed towards platelet-collagen interactions so as to prevent hemostasis in the host during hematophagy. In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen. In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking alpha2beta1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated alpha2beta1-dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor. Moreover, saratin prevented platelet-rich plasma adhesion to fibrillar collagen, a process dependent upon both alpha2beta1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the alpha2 integrin subunit I domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycoprotein Ib and alpha2beta1. These results demonstrate that saratin interferes with integrin alpha2beta1 binding to collagen in addition to inhibiting von Willebrand factor-collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet-collagen interactions.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

Distinct contributions of glycoprotein VI and alpha(2)beta(1) integrin to the induction of platelet protein tyrosine phosphorylation and aggregation

Platelet activation by collagen depends principally on two receptors, alpha(2)beta(1) integrin (GPIa-IIa) and GPVI. During this activation, the nonreceptor protein tyrosine kinase pp72(syk) is rapidly phosphorylated, but the precise contribution of alpha(2)beta(1) integrin and GPVI to signaling for this phosphorylation is not clear. We have recently found that proteolysis of platelet alpha(2)beta(1) integrin by the snake venom metalloproteinase, jararhagin, results in inhibition of collagen-induced platelet aggregation and pp72(syk) phosphorylation. In order to verify whether the treatment of platelets with jararhagin had any effect on GPVI signaling, in this study we stimulated platelets treated with either jararhagin or anti-alpha(2)beta(1) antibody with two GPVI agonists, an antibody to GPVI and convulxin. Platelet shape change and phosphorylation of pp72(syk) by both GPVI agonists was preserved, as was the structure and function of GPVI shown by (125)I-labeled convulxin binding to immunoprecipitated GPVI from jararhagin-treated platelets. In contrast, defective platelet aggregation in response to GPVI agonists occurred in both jararhagin-treated and alpha(2)beta(1)-blocked platelets. This apparent cosignaling role of alpha(2)beta(1) integrin for platelet aggregation suggests the possibility of a topographical association of this integrin with GPVI. We found that both platelet alpha(2)beta(1) integrin and GPVI coimmunoprecipitated with alpha(IIb)beta(3) integrin. Since platelet aggregation requires activation of alpha(IIb)beta(3) integrin, defective aggregation in the absence of alpha(2)beta(1) suggests that this receptor may provide a signaling link between GPVI and alpha(IIb)beta(3). Our study therefore demonstrates that platelet signaling leading to pp72(syk) phosphorylation initiated with GPVI engagement by either convulxin or GPVI antibody does not depend on alpha(2)beta(1) integrin. However, alpha(IIb)beta(3) integrin may, in this model, require functional alpha(2)beta(1) integrin for its activation.     

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.     

Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion

The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.     

The intermediate filament protein vimentin binds specifically to a recombinant integrin alpha2/beta1 cytoplasmic tail complex and co-localizes with native alpha2/beta1 in endothelial cell focal adhesions

Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short alpha and beta cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin alpha2beta1 is a major collagen receptor but to date, only few proteins have been shown to interact with the alpha2 cytoplasmic tail or with the alpha2beta1 complex. In order to identify novel binding partners of a alpha2beta1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-alpha2 and GST-Jun alpha2 bound His-tagged calreticulin while GST-beta1 and GST-Fos beta1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun alpha2/GST-Fos beta1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with alphavbeta3-positive focal contacts. Here, we provide evidence that this interaction also occurs with alpha2beta1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.     

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.     

Distinct structural attributes regulating von Willebrand factor A1 domain interaction with platelet glycoprotein Ibalpha under flow

We have used recombinant von Willebrand factor (vWF) fragments to investigate the properties regulating A1 domain interaction with platelet glycoprotein (GP) Ibalpha. One fragment, rvWF508-704, represented the main portion of domain A1 (mature subunit residues 497-716) within the Cys509-Cys695 disulfide loop. The other, rvWF445-733, included the carboxyl-terminal region of domain D3, preceding A1, and corresponded to the proteolytic fragment originally identified as the GP Ibalpha-binding site (residues 449-728). Conformational changes were induced by reduction and alkylation of the Cys509-Cys695 bond and/or exposure to acidic pH. The cyclic rvWF445-733 fragment exhibited the function of native vWF A1 domain. When immobilized onto a surface, it tethered platelets at shear rates up to 6,300 s-1 mediating low velocity translocation but not stable attachment; in solution, it exhibited limited interaction with GP Ibalpha. In contrast, fragments with perturbed conformation could not tether platelets at high shear rates but promoted stable adhesion at lower shear and bound tightly to GP Ibalpha. Only in the presence of the exogenous modulator, botrocetin, did cyclic rvWF445-733 mediate irreversible adhesion. Thus, conformational transitions in the vWF A1 domain may influence differentially the efficiency of bond formation with GP Ibalpha and the stability of binding.     

Platelet glycoprotein Ib beta/IX mediates glycoprotein Ib alpha localization to membrane lipid domain critical for von Willebrand factor interaction at high shear

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ibα, GP Ibβ, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ibβ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ibβ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ibα disulfide linkage with GP Ibβ markedly decreased the amount of the GEM-associated GP Ibα without altering the GEM association of GP Ibβ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ibα interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ibβ/GP IX mediates the disulfide-linked GP Ibα localization to the GEMs, which is critical for vWf interaction at high shear.     

Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen.

By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site. The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate. The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It inhibited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination. The recombinant polypeptide also inhibited the binding of 125I-vWF to immobilized type I and III collagen. Inhibition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively. Fifty % inhibition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318. Although rGpIb alpha Q221-L318 inhibited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces. Reduction of the disulfide bond between C248 and C264 abolished activity. 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF. These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264. The binding of this recombinant polypeptide to vWF multimers inhibits vWF interaction with two important substrates, platelet GpIb/IX and collagen.     

Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.     

Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.     

Arylamide derivatives as allosteric inhibitors of the integrin alpha2beta1/type I collagen interaction

We herein report a group of allosteric inhibitors of integrin alpha(2)beta(1) based on an arylamide scaffold. Compound 4 showed an IC(50) of 4.80 microM in disrupting integrin I-domain/collagen binding in an ELISA. These arylamide compounds are able to block collagen binding to integrin alpha(2)beta(1) on the platelet surface. Further we find that compound 4 recognizes a hydrophobic cleft on the side of the alpha(2) I-domain, suggesting an alternative targeting site for drug development.     

Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites

We have used purified proteolytic fragments of von Willebrand factor (vWF) to characterize three related functional sites of the molecule that support interaction with platelet glycoprotein Ib, collagen, and heparin. A fragment of 116 kDa was found to be dimeric and consisted of disulfide-linked subunits which, after reduction and alkylation, corresponded to the previously described 52/48-kDa fragment extending from residue 449 to 728. Fragment III-T2, also a dimer, was composed of two pairs of disulfide-linked subunits, two 35-kDa heavy chains (residues 273-511) and two 10-kDa light chains (residues 674-728). The 116-kDa fragment, but not the constituent 52/48-kDa subunit, supported ristocetin-induced platelet aggregation and retained 20% (on a molar basis) of the ristocetin cofactor activity of native vWF; fragment III-T2 retained less than 5% activity. All three fragments, however, inhibited vWF interaction with glycoprotein Ib. Both 116-kDa and 52/48-kDa fragments inhibited vWF binding to heparin with similar potency, while fragment III-T2 had no effect in this regard. Only the 116-kDa fragment inhibited vWF binding to collagen. These results indicate that dimeric fragments containing two glycoprotein Ib-binding sites possess the minimal valency sufficient to support ristocetin-induced aggregation. The sequence comprising residues 512-673, missing in fragment III-T2, is necessary for binding to heparin and collagen and may be crucial for anchoring vWF to the subendothelium. Immunochemical and functional data suggest that the same sequence, although not essential for interaction with glycoprotein Ib, may influence the activity of the glycoprotein Ib-binding site. Only binding to collagen has absolute requirement for intact disulfide bonds. Thus, the three functional sites contained in the 116-kDa domain of vWF are structurally distinct.     

CD40 ligand binds to alpha5beta1 integrin and triggers cell signaling

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.     

Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom of Calloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against alpha(2)beta(1) integrin. This finding suggested that clustering of alpha(2)beta(1) integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of alpha(2)beta(1) integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta(1)-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both alpha(2)beta(1) integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.     

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.     

Modulation of platelet function through adhesion receptors. A dual role for glycoprotein IIb-IIIa (integrin alpha IIb beta 3) mediated by fibrinogen and glycoprotein Ib-von Willebrand factor.

We have found that the form of glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) expressed on nonstimulated platelets is a functional receptor that mediates selective and irreversible adhesion to immobilized fibrinogen. This occurs even in the presence of the elevated intracellular cAMP levels induced by prostaglandin E1 or after inhibition of protein kinase C activity by sphingosine. In the absence of inhibitors, platelets adhering to fibrinogen through GP IIb-IIIa become fully activated and aggregate with one another. Immobilized von Willebrand factor (vWF), in contrast, is recognized by nonstimulated platelets through another receptor, GP Ib. This interaction leads to a change in the ligand recognition specificity of GP IIb-IIIa that can then bind to immobilized vWF and mediate irreversible platelet adhesion and aggregation; this process, however, is inhibited by elevated intracellular cAMP levels or blockade of protein kinase C activity. Therefore, GP Ib and GP IIb-IIIa induce platelet activation through the selective recognition of immobilized vWF and fibrinogen, respectively, in the absence of exogenous agonists. Moreover, "nonactivated" and "activated" GP IIb-IIIa exhibits distinctly different reactivity toward surface-bound vWF, and the functional switch can be induced by the binding of vWF to GP Ib. These findings demonstrate the modulation of platelet function by two different adhesion receptors, GP Ib and GP IIb-IIIa, as well as the distinct dual role of the latter as the necessary common mediator of irreversible adhesion and aggregation on both fibrinogen and vWF.