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. 2014 Dec 31;9(12):e115306.
doi: 10.1371/journal.pone.0115306. eCollection 2014.

Mice lacking the SLAM family member CD84 display unaltered platelet function in hemostasis and thrombosis

Sebastian Hofmann  1 , Attila Braun  1 , Rastislav Pozgaj  1 , Martina Morowski  1 , Timo Vögtle  1 , Bernhard Nieswandt  1
Affiliations

Mice lacking the SLAM family member CD84 display unaltered platelet function in hemostasis and thrombosis

Sebastian Hofmann et al. PLoS One. .

Abstract

Background: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation--platelet adhesion and aggregation--have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.

Objective: The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.

Methods and results: We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84(-/-) platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84(-/-) mice. In vivo, Cd84(-/-) mice exhibited unaltered hemostatic function and arterial thrombus formation.

Conclusion: These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CD84-deficient mice display normal platelet count and size.
(A) CD84 targeting strategy: This scheme illustrates the detection of wild-type and Cd84−/− (targeted) alleles. Upon homologous recombination, the pWH9 cassette containing a neomycin resistance gene disrupts the Cd84 gene. An external probe (EP) recognizes a sequence downstream of 3′ arm in intron 2. With the pWH9 cassette, a new BamHI restriction site is introduced, enabling the determination of a wild-type and Cd84−/− band by Southern blot analysis. (B) Analysis of CD84 expression in wild-type (Cd84+/+) and Cd84−/− platelets by Western blot. Expression of GPIIIa was used as loading control. (C) Peripheral platelet counts and (D) platelet volume of wild-type and Cd84−/− mice measured with a blood cell counter. (E) Determination of the platelet life span in wild-type and Cd84−/− mice. Mice were injected with a DyLight 488-conjugated anti-GPIX Ig derivate to label platelets in vivo. Results are % of fluorescently labeled platelets at the indicated days after injection as determined by flow cytometry. Values are mean ± SD of 5 mice per group.
Figure 2
Figure 2. Normal αIIbβ3 activation, α-granule release and aggregation response of Cd84−/− platelets.
(A) Flow cytometric analysis of integrin αIIbβ3 activation (upper panel) and degranulation-dependent P-selectin exposure (lower panel) in response to the indicated agonists in wild-type and Cd84−/− platelets. Results are mean fluorescence intensities (MFI) ± SD of 4 mice per group and are representative of 4 individual experiments. CRP: collagen-related peptide, CVX: convulxin, and RC: rhodocytin. (B) Washed platelets from wild-type (black line) and Cd84−/− (gray line) mice were activated with the indicated agonist concentrations and light transmission was recorded on a Fibrintimer 4-channel aggregometer. ADP measurements were performed in prp. Representative aggregation traces of at least 3 individual experiments are depicted (for RC: n = 4 mice per group).
Figure 3
Figure 3. Normal integrin outside-in signaling in Cd84−/− platelets.
(A) Washed platelets of wild-type and Cd84−/− mice were allowed to spread on fibrinogen (100 µg/mL) for 30 min after stimulation with 0.01 U/mL thrombin. Statistical evaluation of the percentage of spread platelets at different spreading stages and representative differential interference contrast (DIC) images of 2 individual experiments. Spreading stages: 1: roundish, 2: only filopodia, 3: filopodia and lamellipodia, 4: fully spread (scale bar 5 µm). (B) Clot retraction of Cd84+/+ and Cd84−/− platelets in prp upon activation with 5 U/mL thrombin in the presence of 20 mM CaCl2 at the indicated time points (n = 6, left) and extruded serum after clot formation (right). Values are mean ± SD.
Figure 4
Figure 4. Normal adhesion and aggregate formation of Cd84−/− platelets on collagen under flow.
(A) Whole blood from Cd84+/+ or Cd84 −/− mice was perfused over a collagen-coated surface (0.2 mg/mL) at a shear rate of 1700 s−1. Representative phase contrast images of aggregate formation on collagen after 4 minutes of perfusion time (scale bar 50 µm) (left) and mean surface coverage (right). (B) Representative fluorescence images on aggregate formation on collagen after 4 minutes of perfusion time (left) and relative thrombus volume expressed as integrated fluorescence intensity (IFI) ± SD of n = 5 mice per group (right).
Figure 5
Figure 5. Unaltered thrombotic and hemostatic function in Cd84-deficient mice.
(A) The abdominal aorta was injured by firm compression with a forceps and blood flow was monitored for 30 min. Each symbol represents one animal. (B) Small mesenteric arterioles were injured by topical application of FeCl3 and occlusive thrombus formation was monitored using intravital microscopy. Each symbol represents one mesenteric arteriole. The horizontal dotted line indicates the mean time to vessel occlusion. (C) Representative images of the FeCl3-induced injury model of mesenteric arterioles in Cd84+/+ and Cd84− /− mice, asterisk indicates stable occlusion of the vessel. (D) 1 mm tail tip was amputated and tail bleeding times of Cd84+/+ and Cd84−/− mice were monitored. Each symbol represents one animal. The horizontal dotted line indicates the mean time to vessel occlusion.
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Grants and funding

This work was supported by the German Research Foundation (SFB 688 to BN) and the Rudolf Virchow Center. This publication was funded by the German Research Foundation (DFG) and the University of Würzburg in the funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.