Evidence against a direct role of the integrin α2β1 in collagen‐induced tyrosine phosphorylation in human platelets

Abstract

In the present study we have investigated whether the collagen receptor a2b1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with a2b1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the b1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of a2b1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen-related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of a2b1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than a2b1 as previously thought. These observations show that the integrin a2b1 is not required for regulation of tyrosine phosphorylation by collagen.Keywords: collagen; GPIa-IIa; GPVI; platelets; tyrosine phosphorylation.When the subendothelium lining is damaged, platelets adhere to newly exposed collagen fibres and undergo activation leading to thromboxane A 2 formation, aminophospholipid exposure, granule secretion and platelet aggregation. Several surface glycoproteins have been implicated as collagen receptors, including glycoprotein (GP) IV (also known as CD36), GPVI, a recently cloned protein of 65 kDa protein, a 85/90 kDa protein and the integrin a2b1. GPVI and a2b1 are believed to play pivotal roles in the interaction of platelets with collagen as patients deficient in the expression of either protein have impaired collagen±platelet interactions and mild bleeding disorders. The consensus is that the integrin a2b1 plays a critical role in adhesion, whilst GPVI is essential for activation. There is evidence for minor roles of other receptors in these responses (reviewed in [1±3]).GPVI is associated with the Fc receptor g-chain (FcR g-chain) on the platelet surface [4,5], and crosslinking leads to phosphorylation of the FcR g-chain on a sequence known as an immunoreceptor tyrosine-based activation motif (ITAM) [6], probably by a Src kinase [7,8]. This enables binding and activation of the tyrosine kinase Syk, starting a chain of events that leads to t...

Platelet-collagen interaction: is GPVI the central receptor?

“…Surprisingly, they reported that the peptide has no effect on functional responses and tyrosine phosphorylation in platelet suspensions despite being able to bind to the integrin under these conditions. 106 Hers et al 94 made a similar conclusion through examination of the actions of a number of ␣ 2 ␤ 1 -specific antibodies in platelet suspensions, many of which have been shown to activate the integrin in other systems.…”

“…While some authors reported that inhibition of ␣ 2 ␤ 1 markedly reduced or abolished adhesion and aggregate formation in stasis and flow, [88][89][90] others found only minor effects of such treatment on adhesion to collagen [91][92][93] and collagen-induced aggregation. 94 Several factors are likely to have contributed to these differences, including the nature of the response under investigation; the experimental conditions, notably the presence or absence of plasma; and the preparation of collagen. These are each discussed in further detail in this section.…”

“…Additionally, the mode of ␣ 2 ␤ 1 inhibition may have influenced the results. For example, a number of anti-␣ 2 ␤ 1 agents have also been reported to weakly inhibit platelet activation by GPVI-specific agonists such as convulxin and CRP, 13,89,94 suggesting either an inhibitory effect of the integrin on GPVI-dependent signals or a nonspecific action. Perhaps the clearest indication of a nonspecific inhibitory effect of anti-␣ 2 ␤ 1 treatment came from studies with the snake venom toxin rhodocytin, a powerful platelet agonist.…”

Rac, a small guanosine triphosphate–binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin α2β1

“…However, as described above for platelet aggregation, the presence of integrin ␣ 2 ␤ 1 -mediated activation signals has been challenged by others. 1,11 Thus, we sought to characterize the integrin ␣ 2 ␤ 1 -mediated signals that lead to platelet spreading in this study. Platelets adhere to collagen, then extend projections, and finally form curtainlike extensions between the projections during the process of spreading.…”

Platelet Adhesion to Collagen and Collagen-Related Peptide Under Flow

“…22,37 However, it is not clear how far the integrin is directly responsible for signaling leading to platelet activation, because anti-␣ 2 ␤ 1 antibodies neither block tyrosine phosphorylation nor activation of FAK induced by fibrillar collagen, when aggregation rather then adhesion is dominant. 25,38,39 We also observed that overall tyrosine phosphorylation in control or PP2-treated adherent platelets was unchanged by the 6F1 antibody, even though this antibody decreased adhesion to 17% and 15%, respectively (data not shown). We reported earlier that changes in cGMP levels can influence platelet adhesion to collagen 36 and that PKC is involved in activating the ␣ 2 ␤ 1 integrin and enabling firm adhesion.…”