We have previously assigned an integrin ␣ 2 ␤ 1 -recognition site in collagen I to the sequence, GFOGERGVEG-POGPA (O ‫؍‬ Hyp), corresponding to residues 502-516 of the ␣ 1 (I) chain and located in the fragment ␣ 1 (I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports ␣ 2 ␤ 1 -dependent cell adhesion and exhibits divalent cation-dependent binding of isolated ␣ 2 ␤ 1 and recombinant ␣ 2 A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin ␣ 1 A-domain and supports integrin ␣ 1 ␤ 1 -mediated cell adhesion. Triple-helical GFOGER completely inhibits ␣ 2 A-domain binding to collagens I and IV and ␣ 2 ␤ 1 -dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits ␣ 1 A-domain binding to collagen I and strongly inhibits ␣ 1 ␤ 1 -mediated adhesion of Rugli cells to this collagen but has little effect on either ␣1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for ␣ 2 ␤ 1 and in collagen I for ␣ 1 ␤ 1 . Other high-affinity sites in collagen IV mediate its recognition of ␣ 1 ␤ 1 .The integrins are important receptors mediating both cellcell contact and cellular recognition of the extracellular matrix. They are heterodimers comprising an ␣ and a ␤ chain and are classified according to the identity of the latter (1). Integrin recognition sequences have been identified in a number of matrix proteins. RGDX 1 (where X is one of several possible amino acids) occurs in a wide variety of adhesive glycoproteins and recognizes several of the integrins. In fibronectin, for example, RGD recognizes a number of integrins, including ␣ 5 ␤ 1 , ␣ V ␤ 3 , and ␣ IIb ␤ 3 (2, 3).Integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 are the major integrin collagen receptors (4 -6). Each recognizes a variety of collagens, including collagen I, the most abundant and widely distributed of all the collagens. Recognition of collagen IV by integrin ␣ 1 ␤ 1 has been reported to involve an aspartyl residue at position 461 in the ␣ 1 (IV) collagen chain and an arginyl residue at the same residue position in the ␣ 2 (IV) chain (7).Integrin ␣ 2 ␤ 1 plays an essential role in platelet adhesion to collagens in the blood vessel wall under flow conditions (8). This adhesion depends on collagen being in the triple-helical conformation (9) and is important in hemostasis, but it may also be a crucial initiator of thrombosis. Fragmentation of collagen I has indicated the presence of several ␣ 2 ␤ 1 -binding sites throughout the molecule recognized by platelets (9). In particular, fragment ␣ 1 (I)CB3 2 is as good as the parent collagen in supporting ...

p38 mitogen-activated protein kinase (MAPK) was identified in platelets on the basis of (a) its reactivity with antibodies to C-terminal and N-terminal peptides, and (b) its ability to activate MAPK-activated protein kinase-2, which phosphorylates the small heat shock protein, hsp27. p38 MAPK was activated in platelets by collagen fibers, a collagen-related cross-linked peptide, thrombin, or the thromboxane analogue U46619. A highly specific inhibitor of p38 MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet enzyme in vitro (IC50 approximately 0.5 microM). At similar concentrations it also inhibited agonist-stimulated phosphorylation of hsp27 in platelets, and platelet aggregation and secretion induced by minimal aggregatory concentrations of collagen or U46619, but not thrombin. Inhibition of aggregation was overcome by increasing agonist dose. SB203580 might act by inhibiting thromboxane generation, but this was only inhibited by 10-20% at low agonist concentrations. p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting.

The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences.

The collagen type I-derived fragment ␣ 1 (I)CB3 is known to recognize the platelet collagen receptor integrin ␣ 2 ␤ 1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified ␣ 2 ␤ 1 and the recombinant ␣ 2 A-domain, and their ability to support ␣ 2 ␤ 1 -mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an ␣ 2 ␤ 1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I ␣ 1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of ␣ 2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of ␣ 2 ␤ 1 by the peptide CB3(I)-2 containing the putative ␣ 2 ␤ 1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent ␣ 1 (I)CB3 fragment.Integrins that recognize collagen can modulate cell behavior, including adhesion and spreading, migration, division, metabolism, and the expression of the differentiated phenotype. These important processes are physiologically relevant to growth and development, wound repair, and angiogenesis and in pathological processes such as thrombosis and tumor metastasis.Integrin ␣ 2 ␤ 1 is also an important collagen receptor in hemostasis, where it plays an essential role in the arrest of platelets, under conditions of blood flow, on the collagen fiber surface exposed as a consequence of injury (1-6). Subsequent recognition by the platelet receptor Gp 1 VI of GPP* 2 sequences within the collagen triple helix (7) leads to platelet activation and aggregation with formation of a platelet plug, which serves to stem the loss of blood. Activation of platelets by collagen may also be a cause of thrombosis, especially that associated with rupture of the atherosclerotic plaque, which leads to exposure of underlying collagens (8).Previous fragmentation studies (9) have indicated the presence of a number of integrin ␣ 2 ␤ 1 recognition sites in collagen I, which, with collagen III, represents the main platelet-aggregatory collagen species in the vessel wall and perivascular space (8). In particular, ␣ 2 ␤ 1 -mediated platelet adhesion to fragment ␣ 1 (I)CB3 derived from the ␣ 1 (I) chain of collagen I is as good as to the parent collagen (9, 10). Inhibition studies with short lin...

Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor γ chain, the tyrosine kinase syk, and phospholipase Cγ2 (PLCγ2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support α2β1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin α2β1 . This finding suggests the existence of a platelet receptor other than α2β1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLCγ2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin α2β1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLCγ2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of α2β1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an α2β1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLCγ2. This pathway appears to contribute to platelet activation by collagen.