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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2003 as doi:10.1096/fj.02-0381fje. |
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2ß1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo 1
Department of Biochemistry, CARIM, Maastricht University, Maastricht, The Netherlands;
* Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97078 Würzburg, Germany;
$ Department of Molecular Medicine, Max-Planck Institute for Biochemistry, Martinsried, Germany; and
Department of Pharmacology, University of Heidelberg, 69120 Heidelberg, Germany
2Correspondence: Department of Biochemistry, CARIM, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, the Netherlands. E-mail: jwm.heemskerk{at}bioch.unimaas.nl
SPECIFIC AIM
Platelets vigorously interact with collagen in a damaged vessel wall through glycoprotein VI (GPVI), an immunoglobulin receptor, and integrin 
2ß1, resulting in vaso-occlusive thrombus formation. We earlier demonstrated that GPVI but not 2ß1 integrin is essential in priming platelet interaction with collagen and subsequent aggregation of the platelets. In the present study, we performed flow experiments with whole mouse blood and monitored real-time platelet reactions during their interaction with collagen in order to resolve current discrepancies as to the precise role of either receptor in thrombus formation. We hypothesized that the 2ß1 integrin has a secondary yet relevant role to GPVI in this process. To investigate this, we used genetically modified mice with platelets deficient in either GPVI or 2ß1, as well as mice deficient in Gq whose platelets have lowered reactivity to the autocrine mediators thromboxane A2 (TxA2) and ADP, implicated in aggregation.
PRINCIPAL FINDINGS
1. Integrin 2ß1 supports GPVI-dependent formation of stable aggregates under flow and potentiates platelet procoagulant activity
Perfusion of PPACK anticoagulated whole blood from wild-type mice at high or low wall shear rate (1000 or 150 s-1, respectively) over fibrillar type I collagens resulted in rapid platelet adhesion, followed by formation of aggregates and exposure of procoagulant phosphatidylserine (PS) in part of the platelets. The PS-exposing platelets, usually with a blebbing structure, were identified by staining with Oregon green 488 (OG488) -labeled annexin V (Fig. 1
A, E). To investigate the involvement of GPVI, we used mice deficient in FcR 
-chain as well as mice injected with the anti-GPVI antibody JAQ1; these animals contain platelets virtually absent in GPVI receptors. In either case, only few platelets were deposited on the collagen under flow. These platelets did not show morphological signs of activation and did not aggregate or expose PS (see Fig. 1B, F
). The same results were obtained if wild-type blood was preincubated with Fab fragments of the JAQ1 antibody.
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To create platelets lacking integrin 2ß1, mice carrying a floxed ß1 gene and expressing Cre recombinase were subjected to Mx-Cre-mediated deletion of this ß1 gene. In whole-blood flow experiments (1000 s-1), platelets from these ß1-null mice adhered transiently but still formed aggregates, which had a looser structure than those of wild-type platelets. Strikingly, the ß1-null platelets showed markedly reduced annexin V binding (Fig. 1C, G
). Similar results were obtained when control blood was pretreated with the blocking anti-2ß1 antibody LEN1.
In contrast, high-shear flow experiments with blood from Gq mice resulted in normal platelet–collagen interaction and normal pseudopod and bleb formation, but the aggregates here remained small and were no more than two cell layers thick. Exposure of PS was unchanged (Fig. 1D, H
). This corroborates the evidence that platelets from these mice lack responsiveness to TxA2 and ADP via the TP and P2Y1 receptors, respectively, and thus show deviant autocrine effects. Together, these results confirm and extend the essential role of GPVI in platelet–collagen interaction. They demonstrate that 2ß1 integrin, but not Gq, is required to potentiate GPVI-dependent PS exposure, i.e., procoagulant activity.
2. Integrin 2ß1, but not Gq, contributes to sustained, GPVI-induced Ca2+ responses under flow
To determine the activation state of platelets interacting with collagen under flow, blood was spiked with fluo-3-loaded platelets derived from the same mouse type at a concentration of 5% labeled platelets. Labeled wild-type platelets stably attached to the collagen fibrils and exhibited a potent increase in cytosolic [Ca2+]i. In the absence of functional GPVI, i.e., with blood from FcR -chain deficient mice or JAQ1-injected mice, the few adhering platelets remained continuously low in [Ca2+]i. The same lack of Ca2+ signal generation was seen in control blood preincubated with JAQ1 antibody. In contrast, the absence of 2ß1 integrin (ß1-null mice) resulted in mostly transient adhesion of the fluo-3-labeled platelets accompanied by a reduced Ca2+ signal. Absence of Gq influenced neither the stable adhesion to collagen nor the potent Ca2+ response. These results demonstrate that GPVI is required for the generation of Ca2+ in platelets deposited from the flowing blood, whereas 2ß1 serves to potentiate the GPVI-induced Ca2+ signaling. Gq-dependent stimulation of phospholipase Cß does not appear to be involved in the generation of this Ca2+ signal.
3. Secretion of TxA2 and ADP mediates formation of large platelet aggregates but not the primary GPVI-dependent Ca2+ responses and procoagulant activity under flow
To mimic the effects of Gq abolishment, blood from wild-type mice was treated with aspirin (blocking TxA2 formation) and MRS2179 (a specific P2Y1 antagonist). In flow experiments, this resulted in formation of no more than small aggregates while many of the collagen-adherent platelets were still blebbing and exposed PS. Concurrent inhibition of the second platelet ADP receptor P2Y12 (coupled to Gi) by the potent antagonist AR-C69931MX resulted in small aggregates, but reduced the number of PS-exposing platelets with 
50%. These data suggest that TxA2 and ADP secreted from collagen-activated platelets during flow are essential for the formation of large platelet aggregates but of moderate importance in platelet–collagen interaction and subsequent activation. This hypothesis was confirmed by the observation that treatment of control blood with aspirin/MRS2179/AR-C69931MX did not alter the sustained increase in [Ca2+]i of the adherent platelets. This confirms the idea that GPVI, and not the released autocrine substances, mediates activation of the collagen-bound platelets.
CONCLUSIONS AND SIGNIFICANCE
Collagen-bound platelets trigger the process of thrombus formation, and thus arterial thrombosis, by trapping flowing platelets into aggregates, releasing the autocrine substances TxA2 and ADP and exposing procoagulant PS at their outer surface, which potently stimulates prothrombinase activity and thereby blood coagulation. Our data provide novel, detailed insight into the different roles of GPVI and integrin 2ß1 in these platelet responses. Depletion of GPVI or blocking of this receptor with JAQ1 antibody fragment essentially abolished platelet adhesion to collagen under flow. The few GPVI-deficient or -inactivated platelets that adhered, probably via GPIb binding to von Willebrand factor deposited on the collagen, remained unstimulated. They remained low in [Ca2+]i, did not spread or accrete other platelets, and failed to expose procoagulant PS. Thus, GPVI acts as initiating receptor that induces platelet adhesion as well as subsequent activation and aggregation reactions.
Experiments with blood from ß1-null mice and the blocking anti-2ß1 antibody LEN1 point to a different, complementary role of the integrin receptor. In the absence of functional 2ß1, platelets adhered less firmly to collagen under flow, whereas the formed aggregates had a looser structure. This coincided with a decreased Ca2+ response and a marked reduction in PS exposure in comparison to control conditions with wild-type platelets. Collectively, these data suggest that, after activation by GPVI, the integrin 2ß1 serves to facilitate the various GPVI-dependent signaling events by stabilizing platelet adhesion to collagen, causing prolonged and magnified platelet activation. Both receptors thus intimately cooperate in the collagen-induced thrombus formation: GPVI as an initiating and signaling receptor, and active integrin 2ß1 as a high-affinity adhesive receptor allowing GPVI to signal.
It was also found that Gq-deficient platelets normally adhere to collagen under high-shear conditions and elicit normal rises in [Ca2+]i, followed by PS exposure. This demonstrates for the first time that Gq is not involved, either directly or through TxA2 or ADP, in the direct platelet interaction with collagen. However, as Gq-deficient platelets failed to form large aggregates, it is inferred that release of TxA2 and/or ADP under flow (mediated by GPVI) is required to trap flowing platelets to assemble into aggregates. Indeed, similar adhesion and aggregation as with Gq-deficient platelets was obtained with wild-type platelets, when the TxA2 formation was inhibited and the ADP receptor P2Y1 was blocked using MRS2179. Accordingly, the autocrine mediators mainly promote aggregation.
Taken together, our data provide novel, detailed insight into the different roles of two important receptors that are implicated in normal hemostasis and arterial thrombosis. They can resolve the apparent discrepancy in the literature whether the 2ß1 integrin is or is not primarily involved in platelet–collagen interaction. In the schematic model (Fig. 2
), binding and activation via GPVI prime for integrin 2ß1-mediated adhesion. Conversely, the activated integrin (also perhaps by other agonists) stabilizes the platelets on collagen, so that GPVI can complete its signaling task. The data thus provide a new rationale of why agents inhibiting 2ß1, perhaps in combination with anti-GPVI effects, can be useful for platelet-directed anti-thrombotic therapies. In addition, they provide a first example of interplay of immunoglobulin receptors (GPVI) and integrins (2ß1) in cellular activity. Given the importance of either receptor family in cell biology, similar cross-talk may be instrumental in the functioning of other cellular types.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0381fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0381fje 
3 Both authors contributed equally to this paper.
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