The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa

We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique alpha subunit, recognized by the antibodies, and a common beta subunit, each of approximately 140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, II, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains.     

A new platelet receptor specific to type III collagen. Type III collagen-binding protein

Platelet interaction with type III collagen is mediated by several platelet receptors that recognize specific sequences in collagen. We previously described an octapeptide KP*GEP*GPK within the alpha(1)III-CB4 fragment that binds to platelets and specifically inhibits platelet aggregation induced by type III collagen. In this study, we demonstrated that the octapeptide prevented platelet contact and spreading on type III collagen and subendothelium under static and flow conditions. Platelets adhered to the immobilized octapeptide, and anti-bodies directed against other platelet collagen receptors (glycoprotein (GP) Ia/IIa, GP IV, p65, p47) did not impair this adhesion. The platelet octapeptide receptor was identified by ligand blotting as a protein doublet with molecular masses of 68 and 72 kDa and does not correspond to any other already known platelet collagen receptors (GP Ia, GP IV GP VI, and p65). Our results indicate that a specific type III collagen receptor, expressed on the platelet surface, is involved in the first stages of platelet type III collagen interaction.     

Isolation and characterization of a platelet surface collagen binding complex related to VLA-2

A heterodimeric, Mg++-dependent, collagen binding protein has been isolated from platelet membranes. Electrophoretic properties and monoclonal antibody reactivity indicate that the heavy chain of the complex is platelet membrane glycoprotein Ia and that the light chain is glycoprotein IIa. Furthermore, the receptor appears to be identical with the recently defined VLA-2 complex found on activated T-lymphocytes, platelets and other cells. When incorporated into liposomes, the purified complex mediates the Mg++-dependent adhesion of the liposomes to collagen substrates. These observations suggest that the VLA-2 complex mediates cellular adhesion to collagen in platelets and possibly in other cells.     

Functional analysis of a recombinant glycoprotein Ia/IIa (Integrin alpha(2)beta(1)) I domain that inhibits platelet adhesion to collagen and endothelial matrix under flow conditions

The interaction of platelets with collagen plays an important role in primary hemostasis. Glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) is a major platelet receptor for collagen. The binding site for collagen has been mapped to the I domain within the alpha(2) subunit (GPIa). In order to assess the role of the alpha(2)-I domain structure in GPIa/IIa binding to collagen, a recombinant I domain (amino acids 126-337) was expressed in Escherichia coli. The alpha(2)-I protein bound human types I and III collagen in a saturable and divalent cation-dependent manner and was blocked by the alpha(2)beta(1) function blocking antibody 6F1. The alpha(2)-I protein inhibited collagen-induced platelet aggregation (IC(50) = 600 nM). Unexpectedly, 6F1, an antibody that fails to inhibit platelet aggregation in platelet-rich plasma, blocked the inhibitory effect of the alpha(2)-I protein. The alpha(2)-I protein was able to prevent platelet adhesion to a collagen surface exposed to flowing blood under low shear stress. Interestingly, it inhibited platelet adhesion to extracellular matrix at high shear stress. These results, taken together, provide firm evidence that GPIa/IIa directly mediates the first contact of platelets with collagen under both stirring and flow conditions.     

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.     

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.     

Platelet membrane glycoproteins and their function: an overview

The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.     

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.     

Inhibition of collagen-induced platelet reactivity by DGEA peptide

Direct interactions between collagen, the most thrombogenic component of the extracellular matrix, and platelet surface membrane receptors mediate platelet adhesion and induce platelet activation and aggregation. In this process two glycoproteins are crucial: integrin alpha2beta1, an adhesive receptor, and GPVI, which is especially responsible for signal transduction. Specific antagonists of the collagen receptors are useful tools for investigating the complexity of platelet-collagen interactions. In this work we assessed the usefulness of DGEA peptide (Asp-Gly-Glu-Ala), the shortest collagen type I-derived motif recognised by the collagen-binding integrin alpha2beta1, as a potential antagonist of collagen receptors. We examined platelet function using several methods including platelet adhesion under static conditions, platelet function analyser PFA-100TM, whole blood electric impedance aggregometry (WBEA) and flow cytometry. We found that DGEA significantly inhibited adhesion, aggregation and release reaction of collagen activated blood platelets. The inhibitory effect of DGEA on static platelet adhesion reached sub-maximal values at millimolar inhibitor concentrations, whereas the specific blocker of alpha2beta1 - monoclonal antibodies Gi9, when used at saturating concentrations, had only a moderate inhibitory effect on platelet adhesion. Considering that 25-30% of total collagen binding to alpha2beta1 is specific, we conclude that DGEA is a strong antagonist interfering with a variety of collagen-platelet interactions, and it can be recognised not only by the primary platelet adhesion receptor alpha2beta1 but also by other collagen receptors.     

The platelet receptor GPVI mediates both adhesion and signaling responses to collagen in a receptor density-dependent fashion.

The platelet response to collagen is a primary event in hemostasis and thrombosis, but the precise roles of the numerous identified platelet collagen receptors remain incompletely defined. Attention has recently focused on glycoprotein VI (GPVI), a receptor that is expressed on platelets in association with a signaling adapter, the Fc receptor gamma chain (Fc Rgamma). Genetic and pharmacologic loss of GPVI function results in loss of collagen signaling in platelets, but studies to date have failed to demonstrate that GPVI-Fc Rgamma expression is sufficient to confer collagen signaling responses. These results have led to the hypothesis that collagen responses mediated by GPVI-Fc Rgamma may require the collagen-binding integrin alpha2beta1 as a co-receptor, but this model has not been supported by a recent study of mouse platelets lacking alpha2beta1. In the present study we have used a novel anti-GPVI monoclonal antibody to measure the level of GPVI on human platelets and to guide the development of GPVI-expressing cell lines to assess the role of GPVI in mediating platelet collagen responses. GPVI receptor density on human platelets appears tightly regulated, is independent from the level of alpha2beta1 expression, and significantly exceeds that on previously characterized GPVI-expressing RBL-2H3 cells. Using newly generated GPVI-expressing RBL-2H3 cells with receptor densities equivalent to that on human platelets, we demonstrate that GPVI expression confers both adhesive and signaling responses to collagen in a graded fashion that is proportional to the GPVI receptor density. These results resolve some of the conflicting data regarding GPVI-collagen interactions and demonstrate that 1) GPVI-Fc Rgamma expression is sufficient to confer both adhesion and signaling responses to collagen, and 2) GPVI-mediated collagen responses are receptor density-dependent at the receptor levels expressed on human platelets.     

Rhodocytin induces platelet aggregation by interacting with glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). Involvement of GPIa/IIa-associated src and protein tyrosine phosphorylation

Although glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) has established its role as a collagen receptor, it remains unclear whether GPIa/IIa mediates activation signals. In this study, we show that rhodocytin, purified from the Calloselasma rhodostoma venom, induces platelet aggregation, which can be blocked by anti-GPIa monoclonal antibodies. Studies with rhodocytin-coupled beads and liposomes loaded with recombinant GPIa/IIa demonstrated that rhodocytin directly binds to GPIa/IIa independently of divalent cations. In vitro kinase assays and Western blotting of GPIa immunoprecipitates revealed that Src and Lyn constitutively associate with GPIa/IIa and that Src activity increases transiently after rhodocytin stimulation. Src specifically associates with p130 Crk-associated substrate (Cas) in a manner dependent upon Cas phosphorylation, suggesting that Src is responsible for Cas tyrosine phosphorylation. While all these phenomena occur early after rhodocytin stimulation in a cAMP-resistant manner, tyrosine phosphorylation of Syk and phospholipase Cgamma2, intracellular Ca(2+) mobilization, and platelet aggregation occur later in a cAMP-sensitive manner. Cytochalasin D, which interferes with actin polymerization and blocks receptor clustering, inhibits all the rhodocytin-mediated signals we examined in this study. We suggest that rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated Src, which then mediates downstream activation signals.     

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.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

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.     

Crystal structure of EMS16 in complex with the integrin alpha2-I domain

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin alpha2beta1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin alpha2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Angstrom resolution reveals that the collagen-binding site of the alpha2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the alpha2-I domain. The bound alpha2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the alpha2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the alpha2-I domain, suggesting that EMS16 may have the potential to bind specifically to the alpha2-I domain in a metal ion-independent fashion.     

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper), stimulates platelets by binding to α2β1 integrin and glycoprotein Ib, activating Syk and phospholipase Cγ 2, but does not involve the glycoprotein VI/Fc receptor γ chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.     

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.     

Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen.

The integrin alpha(2)-subunit was ablated in mice by targeted deletion of the ITGA2 gene. alpha(2)-Deficient animals develop normally, are fertile, and reproduce. Surprisingly, no obvious anatomical or histological differences were observed in mutant mice. Besides its significance in tissue morphogenesis, integrin alpha(2)beta(1) has been reported to play a major role in hemostasis by mediating platelet adhesion and activation on subendothelial collagen. To define its role in hemostasis, alpha(2)-deficient platelets were analyzed for their capacity to adhere to and aggregate in response to fibrillar or soluble collagen type I. We show that aggregation of alpha(2)-deficient platelets to fibrillar collagen is delayed but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, alpha(2)-deficient platelets normally adhere to fibrillar collagen. However, in the presence of an antibody against GPVI (activating platelet collagen receptor), adhesion of alpha(2)-deficient but not wild type platelets is abrogated. These results demonstrate that integrin alpha(2)beta(1) significantly contributes to platelet adhesion to (fibrillar) collagen, which is further confirmed by the abolished adhesion of alpha(2)-deficient platelets to soluble collagen. Thus, alpha(2)beta(1) plays a supportive rather than an essential role in platelet-collagen interactions. These results are in agreement with the observation that alpha(2)beta(1)-deficient animals suffer no bleeding anomalies.     

Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2

Collagen plays a critical role in hemostasis by promoting adhesion and activation of platelets at sites of vessel injury. In the present model of platelet-collagen interaction, adhesion is mediated via the inside-out regulation of integrin alpha2beta1 and activation through the glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex. The present study extends this model by demonstrating that engagement of alpha2beta1 by an integrin-specific sequence from within collagen or by collagen itself generates tyrosine kinase-based intracellular signals that lead to formation of filopodia and lamellipodia in the absence of the GPVI-FcR gamma-chain complex. The same events do not occur in platelet suspensions. alpha2beta1 activation of adherent platelets stimulates tyrosine phosphorylation of many of the proteins in the GPVI-FcR gamma-chain cascade, including Src, Syk, SLP-76, and PLCgamma2 as well as plasma membrane calcium ATPase and focal adhesion kinase. alpha2beta1-mediated spreading is dramatically inhibited in the presence of the Src kinase inhibitor PP2 and in PLCgamma2-deficient platelets. Spreading is abolished by chelation of intracellular Ca2+. Demonstration that adhesion of platelets to collagen via alpha2beta1 generates intracellular signals provides a new insight into the mechanisms that control thrombus formation and may explain the unstable nature of beta1-deficient thrombi and why loss of the GPVI-FcR gamma-chain complex has a relatively minor effect on bleeding.     

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)