C.Brakebusch and W.Bergmeier contributed equally to this workPlatelet adhesion on and activation by components of the extracellular matrix are crucial to arrest posttraumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin a2b1 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 b1 integrin on platelets has no signi®cant effect on the bleeding time in mice. Aggregation of b1-null platelets to native ®brillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, b1-null platelets adhere to ®brillar, but not soluble collagen under static as well as low (150 s ±1 ) and high (1000 s ±1 ) shear ow conditions, probably through binding of aIIbb3 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 ®brillar as well as soluble collagen. These data show that GPVI plays the central role in platelet±collagen interactions by activating different adhesive receptors, including a2b1 integrin, which strengthens adhesion without being essential. Keywords: collagen/Cre/loxP/GPVI/a2b1 integrin/ platelets IntroductionDamage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers adhesion and aggregation of platelets (Weiss, 1975). The consequence of this process is the formation of a thrombus, which prevents blood loss at sites of injury or leads to occlusion and irreversible tissue damage or infarction in diseased vessels. Integrins play a central role in adhesion and aggregation of platelets (Phillips et al., 1991;Shattil et al., 1998). Integrins are heterodimeric transmembrane receptors composed of an a and a b subunit. Resting platelets express their integrins in a low af®nity state. After activation, mediated by other platelet receptors, integrins shift to a high af®nity state and bind their ligands ef®ciently (Phillips et al., 1991;Shattil et al., 1998).The ECM of the vessel walls is rich in collagens, which play essential roles in thrombus formation by providing a substrate for platelet adhesion and by activating platelets (Baumgartner, 1977). Besides GPIb-V-IX and aIIbb3 integrin, which interact indirectly with collagen via von Willebrand factor (vWF) (Savage et al., 1998), a large number of collagen receptors have been identi®ed on platelets, including most importantly a2b1 integrin (Santoro, 1986) and glycoprotein VI (GPVI) (Moroi et al., 1989). A current model of platelet adhesion to collagen suggests that the GPIb±vWF interaction mediates initial tethering of platelets at high shear, followed by a2b1 integrin-mediated ®rm adhesion, which halts platelet translocation and allows collagen interactions with GPVI, ®nally resulting in platelet activation and thrombus growth (Sixma et...

The platelet glycoproteins (GPs) Ib, integrin ␣ 2 ␤ 1 , and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s ؊1 ), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb␣ (12G1 Fab 2 ) and ␣ 2 ␤ 1 (6F1 mAb or GFOGER-GPP peptide).GPVI was found to be crucial for aggregate formation, Ca 2؉ signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting ␣ 2 ␤ 1 revealed its involvement in regulating Ca 2؉ signaling, PS exposure, and aggregate size. Both GPIb␣ and ␣ 2 ␤ 1 contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb␣ or ␣ 2 ␤ 1 . In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with ␣ 2 ␤ 1 mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation. IntroductionThe platelet response to exposed subendothelial matrix is fundamental to thrombosis and hemostasis. Uniquely, collagen, the most abundant vessel wall protein, mediates platelet adhesion and activation, localizing and regulating the hemostatic response at sites of injury. Discovering the molecular mechanisms that control platelet-collagen interaction is crucial for understanding the pathogenesis of arteriothrombotic diseases such as stroke and myocardial infarction. Under high shear rate conditions, the glycoprotein (GP) Ib/V/IX complex allows initial platelet rolling over von Willebrand factor (VWF) bound to subendothelial collagen fibers, and subsequently collagen receptors come into contact with their specific binding sequences in the collagen. For the next step, platelet arrest and activation, firm evidence exists of a role for only 2 receptors, integrin ␣ 2 ␤ 1 and immunoglobulin superfamily member GPVI, despite the apparent redundancy in collagen receptors (for a review, see Siljander PR-M and Farndale RW 1 ).According to the 2-site, 2-step model, high-affinity interaction through ␣ 2 ␤ 1 stops the platelet, allowing low-affinity binding of GPVI, which generates signaling required for the subsequent thrombus formation. Platelet deposition under flow was found to be dependent on GPIb/V/IX and ␣ 2 ␤ 1 , 2-4 whereas no platelet deposition occurred on the GPVI-specific substrate collagen-related peptide (CRP), even under low shear rates. 5 The limited number of studies with human platelets deficient in either GPVI or ␣ 2 ␤ 1 support the 2-site, ...

With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2–loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i , followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, “balloon”-like structures (diameter about 5 μm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i , by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI-evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in G(alpha)q or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen-induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin alpha2beta1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.