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Adhesion of human and mouse platelets to collagen under shear: a unifying model

Jocelyn M. Auger^*,,1, Marijke J. E. Kuijpers, Yotis A. Senis^*, Steve P. Watson^* and Johan W. M. Heemskerk

^* Centre for Cardiovascular Sciences, Division of Medical Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham, UK; and
Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands

¹ Correspondence: Centre for Cardiovascular Sciences, Division of Medical Sciences, Institute of Biomedical Research, Wolfson Dr., The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. E-mail: jma299@bham.ac.uk; jwm.heemskerk{at}bioch.unimass.hl

SPECIFIC AIMS

Our understanding of the mechanisms that underlie platelet adhesion and thrombus formation under arterial shear conditions have altered significantly over the past 25 years. Key roles for the leucine-rich von Willebrand factor (VWF) receptor glycoprotein (GP) Ib-IX-V and the integrin IIbß3 in platelet tethering and aggregate formation are established. However, the precise interplay of the collagen receptors, the immunoglobulin GPVI and the integrin 2ß1, in supporting adhesion and aggregate formation on collagen is unclear. Differences between human and mouse have been proposed. Src kinases play a central role in signaling by the major classes of platelet receptors that underlie adhesion and aggregation, but their contribution to these processes under shear in whole blood has not yet been elucidated.

The present study was undertaken to compare the roles of 2ß1, GPVI, and Src kinases in supporting platelet adhesion, activation, and aggregation at intermediate shear. The mechanisms in human and mouse were compared using the same experimental conditions and identical or equivalent experimental tools, including a novel inhibitor of Src kinases, PD0173952, which is effective in human and mouse plasma.

PRINCIPAL FINDINGS

1. Src kinase inhibition abolishes platelet aggregation and GPVI-dependent markers of platelet activation on collagen under shear
Human or mouse blood was flowed over a collagen-coated surface at a shear rate of 1000 s^–1. In both species, platelet deposition and aggregate formation on the collagen fibers increased with time; by 4 min, platelet aggregates expressing the procoagulant phospholipid phosphatidylserine (PS) could readily be seen on the collagen-coated surface (Fig. 1 Ai, Bi). Treatment of anticoagulated human or mouse blood with the Src kinase inhibitor PD0173952 prevented aggregate formation and PS exposure but significant adhesion of single platelets to the collagen surface remained (Fig. 1Aii, Bii ). Collagen-induced increases in [Ca²⁺]_i in platelets labeled with the calcium-sensitive dye Fluo-3 were completely inhibited in the presence of PD0173952, confirming the effectiveness of the Src kinase inhibitor in abolishing GPVI-induced signaling in both species (not shown).

View larger version (58K): [in this window] [in a new window]   Figure 1. 2ß1 and not IIbß3 mediates Src kinase-independent adhesion to collagen. A) Anticoagulated human and mouse blood was flowed over a collagen-coated surface in a parallel plate flow chamber for 4 min at a shear rate of 1000 s-1. Phase contrast images were recorded (120x120 µm). B) Stably adherent platelets were labeled via perfusion with FITC-conjugated annexin A5. Fluorescent images were recorded (150x150 µm). Blood was pretreated with vehicle (0.1% DMSO), 25 µM PD0173952 (Src kinase inhibitor), 20 µg/mL 6F1 (anti-human 2ß1 mAb) or 20 µg/mL Sam.G4 (anti-mouse 2ß1 mAb), 20 µg/mL Sam.G4 or 20 µg/ml 6F1 and 25 µM PD0173952, 10 µM lotrafiban (IIbß3 antagonist) or 10 µM lotrafiban and 25 µM PD0173952 for 15 min, as indicated above panels. Results are representative of 3–10 experiments. Numbers below images indicate mean surface area coverage as a 5 of control (i) ± SE.

2. Src kinase-independent adhesion is mediated via integrin 2ß1 and not IIbß3
Because complete Src kinase inhibition with PD0173952 did not completely abrogate platelet adhesion to collagen, the roles of 2ß1 and IIbß3 in mediating adhesion under these conditions were evaluated. Monoclonal antibodies (mAbs) 6F1 and Sam.G4 were used to block human and mouse 2ß1, respectively. At optimal concentrations, these mAbs reduced the number and size of aggregates formed on fibrillar collagen under shear at the experimental end point of 4 min, although many single adherent platelets and small-size aggregates remained (Fig. 1Ai, iii ). Both antibodies markedly inhibited PS exposure, but a small number of labeled platelets was still observed (Fig. 1Bi, iii ). This contrasts with the complete blockade of PS exposure observed with Src kinase inhibition (Fig. 1Bii ). In the presence of PD0173952, adhesion of human and mouse platelets was markedly blocked with mAbs 6F1 and Sam.G4, respectively, demonstrating that it was mediated predominantly through 2ß1 (Fig. 1Aiv ). Together, these results indicate that integrin 2ß1 contributes to platelet adhesion to collagen in both species whether Src kinases are active or not.

The IIbß3 antagonist lotrafiban was used to investigate the role of this integrin in adhesion to collagen. Aggregate formation of human and mouse platelets was abolished in the presence of 10 µM lotrafiban (Fig. 1Av ), consistent with the critical role of IIbß3 in mediating thrombus formation. However, there was a high level of adhesion of single platelets in the presence of lotrafiban, which resulted in a small increase in surface coverage relative to controls (Fig. 1Ai, v ). This increase in adhesion to collagen may reflect an increase in the number of platelet-collagen interactions able to take place in the absence of large aggregates, which would shield the collagen surface downstream of flow. The lotrafiban-resistant adhesion was slightly reduced in the presence of PD0173952 (Fig. 1v, vi ) and almost completely inhibited in the presence of an anti-2ß1 blocking antibody (not shown). There was a small increase in PS exposure in the presence of lotrafiban relative to controls (Fig. 1Bi, iv ), paralleling the increase in number of platelets interacting with the collagen fibers. This PS exposure was completely inhibited in the presence of PD0173952 (not shown), confirming that Src kinases are required for this platelet response regardless of IIbß3 activity.

The results demonstrate that integrin IIbß3 is not required for platelet adhesion or activation on a collagen surface. This integrin does not contribute to adhesion in the presence of the Src kinase inhibitor PD0173952 in contrast to the much greater role of integrin 2ß1, highlighting an important difference between the two integrins in supporting adhesion in the absence of Src kinase activity.

3. Real-time analysis of platelet adhesion and activation on collagen
Dynamic information from real-time video imaging was used to further characterize the mechanisms of platelet adhesion to collagen. A large number of individual Fluo-3-loaded platelets were characterized according to the length of time between when a platelet initially interacts with the collagen-coated surface and [Ca²⁺]_i starts to rise. Under human control conditions, 48% of the platelets underwent essentially immediate activation occurring 0.24 ± 0.08 s (mean±SD) after initially interacting with collagen. Most other human platelets (38%) were activated after a delay, with a prolonged but highly variable lag time between adhesion and start of Ca²⁺ signal of 16.3 ± 8.7 s. In the mouse control, 33% of platelets exhibited a fast activation response with a lag time of 0.29 ± 0.19 s; 47% of mouse platelet activation responses were delayed with variable lag times of 15.9 ± 11.9 s, similar to those for human. The majority of human (95%) and mouse (87%) platelets initially interacting with the collagen coated surface stably adhered.

The relative frequency of fast or delayed activation responses was not altered much by treatment with IIbß3 antagonist lotrafiban, confirming that this integrin is not directly involved in platelet-collagen interaction. On the other hand, after blocking Src kinases with PD0173952, the frequency of all activation responses was greatly reduced in human and mouse. The fraction of stably adherent platelets was reduced to 48% for human and 59% for mouse. Similar changes in distribution were seen with GPVI-FcR -chain-deficient mice; in this case, the fraction of platelets with stable adhesion decreased to 41%. Together, these findings indicate that the GPVI/Src kinase signaling pathway is critical for both fast and delayed activation events and that this pathway is responsible for the stable adhesion of a proportion (50%) of the platelets.

Treatment of human and mouse blood with anti-2ß1 mAbs had a significant effect on the relative frequency of responses. The total frequency of fast activation responses was unchanged, showing that 2ß1 plays no role in fast platelet activation. However, the frequency of stably adherent platelets reduced to 59% in human and 40% in mouse, confirming a significant contribution of 2ß1 to stable adhesion. The frequency of delayed activation responses decreased, indicating an additional role for 2ß1 in supporting this response.

CONCLUSIONS AND SIGNIFICANCE

The current study compared roles of the GPVI/FcR -chain complex, Src kinases and integrins 2ß1 and IIbß3 in supporting adhesion and activation of human and mouse platelets on collagen at intermediate shear. These studies were performed in anticoagulated whole blood to mimic the in vivo situation. Direct comparison of human and mouse platelets has become possible because of the availability of specific blocking antibodies and pharmacological inhibitors active in both species, including the IIbß3 antagonist lotrafiban and the novel Src kinase inhibitor PD0173952. Results are considered in light of recent contrasting observations in the literature with regard to the original two-site, two-step model of platelet-collagen interaction.

In human and mouse, inhibition of Src kinases caused a partial reduction in stable platelet adhesion to collagen observed most clearly from analysis of the dynamics of platelet-collagen interaction by real-time fluorescence video microscopy. Approximately half of the platelets adhered stably via GPVI/Src kinases and underwent an essentially immediate increase in [Ca²⁺]_i. The study reveals a GPVI/Src kinase-independent mechanism of stable adhesion active in human and mouse platelets. The latter was not affected by IIbß3 antagonism but almost completely abolished by blocking of 2ß1. Platelets that adhere via this mechanism undergo a delayed increase in [Ca²⁺]_i after adhering to collagen. Normally, individual platelets appear to use either pathway upon interaction with collagen, becoming activated before or after stable adhesion.

Given this heterogeneity of platelet responses, one can envision that many factors would influence the exact sequence of events in individual platelets—for example, the quantity of collagen, the type of GPVI and integrin binding sites on the collagen or the expression of these receptors on the platelet. It is not surprising that, depending on the precise experimental condition, one or the other pathway dominates. For instance, we found that a higher density of collagen coating increases the adhesion of mouse platelets via 2ß1 (unpublished results). The principal mechanism under physiological conditions may vary according to the extent of vessel damage. The two distinct pathways may be a consequence of heterogeneity in the platelet population.

The present study shows that Src kinases play a critical role in platelet activation and aggregate formation at arterial shear that is mediated downstream of GPVI. The study establishes the existence of two routes of platelet adhesion that are dependent on 2ß1 or GPVI/Src kinases. A unifying, two-state model of platelet-collagen interaction is therefore proposed in which GPVI-mediated platelet activation either precedes or follows integrin-mediated platelet adhesion (Fig. 2 ). Both pathways are conserved between human and mouse platelets.

View larger version (23K): [in this window] [in a new window]   Figure 2. Unifying model of platelet adhesion to collagen at arterial shear. Two pathways by which human and mouse platelets firmly adhere to collagen at arterial shear are illustrated. The majority of platelets are initially tethered to collagen via GPIb-IX-V interacting with collagen-bound VWF (left), although a minority of platelets interact directly with collagen independent of VWF/GPIb-IX-V. In the first pathway (upper), signaling from GPVI first leads to activation of integrins 2ß1 and IIbß3. Activated integrins then firmly attach the platelet to collagen directly (2ß1) or via collagen-bound VWF (IIbß3) (right). In the second pathway (lower), platelets first adhere to collagen via integrin 2ß1 before GPVI engages collagen and induces activation. These two pathways are likely to reinforce each other and the events of thrombus formation. Release of secondary mediators (ADP and TxA2) would further potentiate these events (right).

FOOTNOTES

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