Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2 - PubMed (original) (raw)

. 2011 Jul 14;118(2):416-24.

doi: 10.1182/blood-2010-10-312199. Epub 2011 Apr 28.

Sharon A Matthews, Matthew T Harper, Karen Gilio, Judith M E M Cosemans, Christopher M Williams, Maria N Navarro, Deborah A Carter, Johan W M Heemskerk, Michael Leitges, Doreen Cantrell, Alastair W Poole

Affiliations

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

Olga Konopatskaya et al. Blood. 2011.

Abstract

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

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Figures

Figure 1

Figure 1. Agonist-induced PKD activation in platelets is dependent on classic PKC isoforms but independent of novel PKC isoforms

(A) Washed human platelets (2 × 108/mL) were incubated with GFX (10μM), LY333531 (10μM), or vehicle (DMSO) for 10 minutes and subsequently stimulated with CRP (5 μg/mL) or thrombin (1 unit/mL) for 1 minute. (B) Washed human platelets (2 × 108/mL) were incubated with various concentrations of LY333531 or vehicle (DMSO) for 10 minutes and subsequently stimulated with thrombin (1 unit/mL) for 1 minute under stirring conditions. Immunoblots were performed with P-PKD (Ser916), P-PKD (Ser744), or total PKD antibody. One representative experiment of 3 with identical results is shown. (C) Washed platelets (2 × 108/mL) from WT, PKCα−/−, PKCθ−/−, or PKCδ−/− mice, were incubated with various concentrations of CRP or thrombin for 1 minute. Immunoblots were performed with either P-PKD (Ser916) or total PKD antibody. One representative experiment (i) and densitometric analysis of 3 experiments (ii) are shown. For panel Cii, data shown are means ± SEM (n = 3).

Figure 2

Figure 2. PKD expression and activation profile in platelets

Washed platelets from WT, PKD2−/− (A), or PKD2SSAA/SSAA (B) mice (2 × 108/mL) were incubated with CRP (5 μg/mL) or thrombin (1 unit/mL) for 1 minute under stirring conditions. Platelets were then solubilized in Laemmli sample buffer and whole cell lysates were subjected to SDS-PAGE, transferred to PVDF membrane, and immunoblotted with PKD1/2, PKD3, P-PKD (Ser916), or P-PKD (Ser744) antibodies. Data shown are representative of 3 independent experiments.

Figure 3

Figure 3. PKD2 regulates secretion of dense granules in response to CRP and thrombin in platelets

Washed platelets from WT and PKD2SSAA/SSAA mice were stimulated with various concentrations of CRP (A) or thrombin (B) as indicated, and secretion of ATP was assessed by luminometry. Error bars represent SEM, n = 3-6; *P < .05

Figure 4

Figure 4. Biogenesis of dense and α-granules is not regulated by PKD2 activity

Washed platelets from WT or PKD2SSAA/SSAA mice were examined by transmission electron microscopy, and the number of dense granules (black arrows) and α-granules (white arrows) was quantified as described in “Electron microscopy.” (A) Images of platelets from WT (left panel) or PKD2SSAA/SSAA (right panel) mice are representative of 3 independent experiments. Original magnification was 19 000×, and the bar represents 500 nm. (B) Dense and α-granules were counted per fields of view (25-30 fields of view per section, 3 sections per preparation on average) and are shown as means ± SEM, n = 3.

Figure 5

Figure 5. Deficient aggregation responses in platelets from PKD2SSAA/SSAA mice are rescued by exogenously added ADP

Washed platelets from WT and PKD2SSAA/SSAA mice were stimulated with CRP (1.25 μg/mL; A) or thrombin (0.065 units/mL; B) with or without the simultaneous addition of ADP (10μM), and aggregation was assessed turbidimetrically. Aggregation traces are representative of 6 independent experiments with similar results.

Figure 6

Figure 6. PKD2SSAA/SSAA mice display reduced thrombus formation in vitro but have normal tail-bleeding responses

Heparin/PPACK-anticoagulated blood from WT or PKD2SSAA/SSAA mice was passed over collagen (shear rate 1000/s). (Ai) Representative phase-contrast images after 4 minutes of flow. Scale bars represent 20 μm. (ii) Platelet coverage of coated surfaces was quantified and the mean surface area ± standard error of the mean is shown for 3 independent experiments. *P < .05. (B) Mice were anesthetized and a transverse incision made with a scalpel at a position where the diameter of the tail was 2.25-2.5 mm. The tail was immersed in normal saline (37°C) in a hand-held test tube. The time from incision to cessation of bleeding was recorded, and mean times are shown as horizontal bars. No significant difference was seen comparing WT mice with PKD2SSAA/SSAA mice (n = 10-14).

Comment in

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