Trpc6 gain-of-function disease mutation enhances phosphatidylserine exposure in murine platelets

Platelets enhance coagulation by exposing phosphatidylserine (PS) on their cell surface in response to strong agonist activation. Transient receptor potential channels, including TRPC6, have been implicated in the calcium influx central to this process. Here, we characterize the effect of a Trpc6 gain-of-function (GOF) disease-associated, and a dominant negative (DN), mutation on murine platelet activation. Platelets from mice harboring Trpc6E896K/E896K (GOF) and Trpc6DN/DN mutations were subject to in vitro analysis. Trpc6E896K/E896K and Trpc6DN/DN mutant platelets show enhanced and absent calcium influx, respectively, upon addition of the TRPC3/6 agonist GSK1702934A (GSK). GSK was sufficient to induce integrin αIIbβ3 activation, P-selection and PS exposure, talin cleavage, and MLC2 phosphorylation in Trpc6E896K/E896K, but not in wild-type, platelets. Thrombin-induced calcium influx and PS exposure were enhanced, and clot retraction delayed, by GOF TRPC6, while no differences were noted between wild-type and Trpc6DN/DN platelets. In contrast, Erk activation upon GSK treatment was absent in Trpc6DN/DN, and enhanced in Trpc6E896K/E896K, platelets, compared to wild-type. The positive allosteric modulator, TRPC6-PAM-C20, and fluoxetine maintained their ability to enhance and inhibit, respectively, GSK-mediated calcium influx in Trpc6E896K/E896K platelets. The data demonstrate that gain-of-function mutant TRPC6 channel can enhance platelet activation, including PS exposure, while confirming that TRPC6 is not necessary for this process. Furthermore, the results suggest that Trpc6 GOF disease mutants do not simply increase wild-type TRPC6 responses, but can affect pathways not usually modulated by TRPC6 channel activity, displaying a true gain-of-function phenotype.


Introduction
Influx of calcium ions into the cytoplasm is a versatile signaling event utilized by most cell types, including platelets.
[1] Through variation in the location, duration and magnitude of calcium influx, and integration with other signals, calcium signaling can influence a wide range of cellular responses. In platelets, calcium signaling is involved in multiple aspects of activation, including granule release, phosphatidylserine exposure, and shape change.
[2] Several different calcium-permeable ion channels have been implicated in these processes, including TRPC channels.
Canonical transient receptor potential 6 (TRPC6) is a non-specific cation channel member of the transient receptor potential (TRP) superfamily of ion channels. [3][4][5] In addition to acting as a homo-tetramer, TRPC6 can form heteromeric channels with TRPC1, 3 and 7. [6,7] TRPC6 is directly activated by diacylglycerol (DAG), [8] and is thought to act downstream of Gα q coupled receptors, acting as a receptor-operated calcium effector. [9,10] In light of its broad tissue and cell type expression, it is not surprising that TRPC6 is reported to influence multiple physiological and pathophysiological processes. [11][12][13] In humans, TRPC6 mutations are a cause of autosomal dominant focal segmental glomerulosclerosis (FSGS); the majority of these act as gain-of-function. [14,15] However, mice with a Trpc6 E896K allele (corresponding to the human FSGS-associated, GOF E897K mutation) do not develop appreciable glomerular disease. [16] A mechanistic understanding of how TRPC6 mutations lead to kidney disease remains absent.
TRPC6 is abundantly expressed in platelets, [17][18][19][20] but the channel's role in these cell fragments is still incompletely understood. Trpc6 knockout does not affect most agonist stimulated platelet aggregation, integrin activation or degranulation, though there are conflicting data on a role for TRPC6 in response to thromboxane receptor activation. [18,21] TRPC6, together with TRPC3, has been proposed to act as a co-incident detector of thrombin and collagen stimulation to induce phosphatidylserine (PS) exposure.
[20] PS exposure generates a procoagulant surface that enhances thrombin generation. [22] Finally, TRPC6 is required for the exaggerated platelet activation and inflammatory response seen in platelet-specific CFTR knockout mice, but its loss does not affect wild-type platelet function. [23] In the current study, we compared platelets from wild-type, Trpc6 E896K/E896K , and Trpc6 DN/DN mice to further characterize the role of TRPC6 in platelet function. We found that GOF TRPC6 impacts several signaling pathways, including PS exposure, Erk1/2 and MLC2 phosphorylation, talin cleavage and clot retraction, but that most of these pathways are not affected by the loss of TRPC6 channel activity. These studies suggest that TRPC6 GOF mutants do not simply enhance wild-type TRPC6 responses, but can activate effector pathways not normally impacted by TRPC6 channel activity.

Mouse Studies
All animal procedures were approved by the Beth Israel Deaconess Medical Center (BIDMC) Animal Care and Use Committee, and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals carrying mutant Trpc6 alleles, Trpc6 E896K/E896K and Trpc6 DN/DN (introducing an LFW to AAA pore-mutation), were generated at the BIDMC transgenic core using CRISPR/Cas9 with two site-specific guide RNAs (PNA Bio) per locus, Cas9 nickase (PNA Bio), and a single stranded DNA oligo (IDT) carrying the desired mutant sequence. Guide RNA and oligo DNA sequences are listed in Table 1 and 2, respectively. Genomic DNA from founder mice was amplified by PCR and screened by Sanger sequencing. Founders carrying the desired mutation were mated with FVB/NJ animals (Jackson Laboratory). After at least 5 backcrosses with FVB/NJ animals, heterozygous animals were mated to generate homozygous animals and wild-type littermates. Trpc6 E896K/E896K and Trpc6 DN/DN lines were then maintained by mating homozygous animals. Genotyping of the Trpc6 E896K locus was performed using a custom TaqMan SNP assay; the Trpc6 DN locus was genotyped using standard PCR reactions to detect the wild-type and DN alleles. Trpc6 -/mice, previously reported, [24] were obtained from Jackson Laboratory. After crossing the mice with C57BL/6J, heterozygous Trpc6 +/mice were crossed to generate littermate Trpc6 -/and Trpc6 +/+ animals.  Whole blood was obtained via cheek pouch bleeding and collected in EDTA-containing microtainers (BD 365974). Blood counts were analyzed on a Hemavet 950 FS at the BIDMC Small Animal Imaging core facility.
Tail bleeding time was assessed using standard protocols. [19] Briefly, animals were anesthetized with inhaled isoflurane and maintained on a warming blanket. The tail was severed 5 mm from the tip with a sterile scalpel and immediately placed in sterile PBS warmed to 37°C.
The time taken until bleeding stopped, and did not resume for at least 60 seconds, was recorded.
The experiment was terminated if bleeding persisted after 15 minutes.

Platelet Isolation
Mouse platelets were isolated using modified standard procedures.
Platelet rich plasma (PRP) was obtained after centrifugation at 220g for 10 minutes at room temperature. After resting at 37°C for 15 minutes, 1:6 volume ACD solution (75 mM Trisodium citrate, 42 mM citric acid, 136 mM glucose) was added and platelets pelleted at 500g for 10 minutes. Platelets were washed once in 500 µl HEPES-Tyrode buffer with apyrase and PGE 1 , before being resuspended in HEPES-Tyrodes buffer with or without CaCl 2 for use in downstream experiments.

Calcium Imaging
Intracellular calcium levels were assessed using Fura-2 fluorescence ratio measurement (Fura-2 QBT kit R8197, Molecular Devices) on a FlexStation III reader with automated pipetting at the Harvard ICCB-Longwood screening facility. Platelets were resuspended in HEPES-Tyrodes buffer with Fura-2 and 1.2 mM CaCl 2 ; 100 µl of platelet suspension were added to each well of a 96-well plate. Platelets were incubated with Fura-2 for 1hr at 37°C before beginning the assay, and were maintained at 37°C in the FlexStation III reader throughout the experiment. Samples were excited at wavelengths of 340 and 380 nm every 5 s for a total of 330 s, with emissions measured at 510 nm. Baseline fluorescence measurements were obtained for 30 seconds before stimulation by the addition of agonist in 50 µl HEPES-Tyrodes buffer with 1.2 mM CaCl 2 . Final concentrations of agonists/inhibitors were as follows: 10 µM ADP, 3 µM CFTR-inhibitor 172, 10 µM fluoxetine, 50 µM GSK1702934A, 0.5 u/ml thrombin, 10 µM TRPC6-PAM-C20, 5 µM U46619, 10 µM Y-27632. In experiments examining the effects of pre-incubation with inhibitors, drug was added at the start of the Fura-2 incubation.

Flow Cytometry
For phosphatidylserine exposure experiments, platelets were resuspended in HEPES-Tyrode buffer supplemented with 1 mM CaCl 2 , mixed with an equal volume of buffer containing agonist or carrier, and incubated at room temperature for 10 minutes. Agonists and inhibitors were used at the same concentration as for Fura2 imaging experiments, above. 10 µl of platelet suspension was mixed with 40 µl of staining solution (HEPES-Tyrode buffer supplemented with 2.5 mM CaCl2, 1:50 PE rat anti-mouse CD41 antibody (BD Biosciences 561850), and 3:50 FITC Annexin V (BD Biosciences 556419)). Control staining reactions containing one or no fluorophore were performed in parallel. After a 10 minute room-temperature incubation, platelets were diluted with 200 µl of 1.25% paraformaldehyde solution. Cell-surface P-selectin exposure and integrin α IIbβ3 activation was assayed using a two-color mouse platelet activation kit (Emfret Analytics D200) following the supplied protocol. Flow cytometry was performed at the BIDMC flow cytometry core facility on a Beckman Coulter CytoFLEX LX.

Clot Retraction
Clot retraction was assayed following standard protocols.
[27] Citrated PRP was collected as outlined above. 100 µl of PRP and 5 µl of pelleted RBCs (to provide clot contrast) were diluted to 500 µl final volume with HEPES-Tyrode buffer (final 1.5 mM CaCl 2 concentration), supplemented with or without 1 u/ml thrombin, and incubated at room temperature. Tubes were imaged every 15 or 30 minutes for two hours, and two dimensional clot area calculated as a percentage of the total fluid area at each timepoint. A clot "area under the curve" was calculated by summing the clot area for each sample at each timepoint.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism version 9 or later. The statistical tests utilized for each experiment are specified within the corresponding figure legends.

TRPC6 platelet expression and function in Trpc6 mutant animals
Utilizing CRISPR/Cas9, we generated two mouse lines carrying mutations in the Trpc6 gene. As TRPC6 is abundant in platelets, [17][18][19][20] we compared TRPC6 levels in platelets from our mouse lines. Specificity of the anti-TRPC6 antibody was confirmed using platelet lysates from Trpc6 -/animals ( Fig 1A). TRPC6 protein was detected in wild-type, Trpc6 E896K/E896K and Trpc6 DN/DN platelets ( Fig 1B). TRPC6 abundance was slightly lower in KI platelets. Variability in the different molecular weight forms of TRPC6 between genotypes was similar to our previous observations in overexpression systems.
[28] Platelet counts were similar across all three genotypes, as were other aspects of a complete blood count (Table 3). platelets demonstrated a higher peak and a prolonged elevation in the fluorescence ratio (Fig 1C-E). Trpc6 +/DN platelets also failed to respond to GSK, consistent with the mutation's dominant negative phenotype (Fig 1F). Trpc6 +/E896K platelets showed an intermediate Fura-2 response relative to wild-type and Trpc6 E896K/E896K platelets (Fig 1F-H). Platelets from Trpc6 -/animals failed to respond to GSK stimulation, suggesting that TRPC6 is the only major target of this agonist that influences calcium influx in platelets ( Fig 1I). Omitting extracellular calcium completely abolished the agonist effect in Trpc6 E896K/E896K platelets ( Fig 1J). In sum, these results suggest that TRPC6 is required for extracellular calcium influx in response to GSK in platelets, and that the E896K and DN mutant channels display properties similar to what has been reported in in vitro overexpression studies. [6,14] Activation of Gα q coupled receptors is known to activate TRPC6 in heterologous overexpression systems.[8-10] We therefore examined calcium signaling downstream of thrombin, ADP and the thromboxane A 2 receptor agonist, U46619, in Trpc6 mutant platelets. No differences were noted in Fura-2 fluorimetry between wild-type, Trpc6 E896K/E896K and Trpc6 DN/DN platelets in response to ADP (Fig 2A-C). While the peak F340/380 values after thrombin stimulation similarly did not differ between genotypes, the area under the curve was modestly higher in the Trpc6 E896K/E896K platelets compared to Trpc6 DN/DN platelets, but not significantly different between wild-type and Trpc6 DN/DN samples (Fig 2D-F). Responses to U46619 were similar between Trpc6 E896K/E896K and Trpc6 DN/DN platelets (Fig 2G-I). Given this result, wild-type platelets were not tested in keeping with the three R's tenet in animal research. Together, these results suggest that TRPC6 may play a modest role in calcium signaling downstream of thrombin, but is not involved downstream of ADP or U46619.

Effects of Trpc6 mutations on integrin
α IIbβ3 activation and Pselectin exposure Activation of integrin α IIbβ3, and cell surface exposure of P-selectin via degranulation, are two common responses of platelets to agonist stimulation. Previous studies report that Trpc6 deficiency does not affect these responses to various stimuli, including ADP and thrombin, though there is disagreement as to whether thromboxane A2 receptor agonist response is blunted. [18,21] The effect of Trpc6 genotype on integrin α IIbβ3 activation and P-selectin surface expression was assessed by flow cytometry (Fig 3A-B). In line with the lack of effect on ADP and U46619 induced calcium transients (Fig 2), Trpc6 genotype did not affect α IIbβ3 activation or P-selectin surface expression in response to these stimuli (Fig 3A-B). There was also no difference in response to thrombin-mediated activation. GSK alone did not activate wild-type or Trpc6 DN/DN platelets, but did induce low level activation of Trpc6 E896K/E896K platelets.
Combined stimulation with ADP and GSK enhanced P-selectin exposure in wild-type and platelet responses to thrombin alone were similar to each other, the percentage of Annexin positive platelets after combined stimulation with thrombin and GSK was higher in wild-type platelets compared to Trpc6 DN/DN platelets. In our hands, type I collagen or convulxin did not induce PS exposure, or enhance the effect of thrombin on PS exposure (data not shown). As a whole, these results suggest that TRPC6 channel activity is not necessary for PS exposure in response to thrombin, but that Trpc6 gain-of-function mutations may enhance this process.
Indeed, a higher percentage of Trpc6 +/E896K platelets bound Annexin after thrombin stimulation than did wild-type platelets ( Fig 3D).  Fig 4A). Wild-type platelets demonstrated a small, but consistent, increase in P-Erk1/2 levels 5 minutes after stimulation with GSK ( Fig 4B, C). This response was absent in Trpc6 DN/DN platelets, but augmented in Trpc6 E896K/E896K platelets. GSK-mediated Erk activation was dependent on the presence of extracellular calcium ( Fig 4D). Erk activation therefore appears to be a downstream target of TRPC6 activation in platelets. These results confirm our previous studies in heterologous overexpression systems that TRPC6 gain-of-function mutants enhance activation of the Erk1/2 signaling pathway.

Gain-of-function TRPC6 activation induces talin cleavage and delays clot retraction
Phosphatidylserine-exposed platelets tend to inactivate integrin α IIbβ3, in part through the calcium-dependent cleavage of the focal adhesion protein, talin. [35,36] We found that GSK stimulation of Trpc6 E896K/E896K platelets, but not wild-type or Trpc6 DN/DN platelets, induced talin cleavage as assessed by western blot (Fig 5A). This response was seen within a minute after stimulation of Trpc6 E896K/E896K platelets, but remained absent even after 30 minutes of stimulation of wild-type platelets ( Fig 5B).
Cleavage of platelet focal adhesion proteins, including talin, has been associated with relaxed fibrin clot retraction.
[36] There are conflicting reports on the effect of Trpc6 knockout on clot retraction and tail bleeding time. [18,19,21] In our studies, PRP from Trpc6 E896K/E896K animals demonstrated delayed clot retraction compared to PRP from other genotypes, both in the absence of exogenous stimulus, and after thrombin addition (Fig 5C,D). No differences were seen between wild-type and Trpc6 DN/DN genotypes. In contrast to the clot retraction kinetics, tail bleeding time did not appreciably differ between Trpc6 genotypes ( Fig 5E).

Pharmacological modulation of TRPC6-mediated calcium influx and platelet activation
A positive allosteric modulator of TRPC6, TRPC6-PAM-C20 (C20), has been reported to enhance the response of TRPC6 to stimulation by OAG or GSK in human platelets.
[37] We examined whether C20 has a similar effect on murine TRPC6, and whether it might further enhance mutant TRPC6 E896K activity. In wild-type platelets, C20 enhanced GSK-induced calcium influx, but had no effect on its own (Fig 6A-C). This effect was also seen in Trpc6 E896K/E896K platelets, arguing that the positive allosteric effect of C20 acts via a different, and additive, mechanism to that of the gain-of-function E896K mutation.
We hoped to examine how C20 affects TRPC6-dependent phosphatidylserine exposure next.
However, C20 inhibits thrombin-induced Annexin V staining in Trpc6 DN/DN platelets (Fig 6D), suggesting it has TRPC6-independent effects, and preventing interpretation of its effects on TRPC6-dependent PS exposure.
Inhibitors of acid sphingomyelinases, including fluoxetine, are reported to inhibit TRPC6 channel activity [38][39][40][41]. We confirmed that pre-incubation of platelets with fluoxetine inhibits GSK induced calcium influx in wild-type and Trpc6 E896K/E896K platelets ( Fig 7A). In contrast, exposure to fluoxetine did not alter ADP-induced calcium transients ( Fig 7B). The action of fluoxetine is likely indirect, as addition of fluoxetine one minute before GSK had a minimal effect on Fura2 fluorescence, as compared to a one hour pre-incubation ( Loss of CFTR is reported to enhance agonist-mediated platelet activation in a TRPC6-dependent manner [23], while genetic or pharmacologic interference of Rho-associated coiled-coil serine/threonine kinase 1 (ROCK1) enhances platelet PS exposure [42]. We therefore examined whether pharmacological inhibition of CFTR and ROCK affects TRPC6-mediated platelet activities (Fig 8). CFTR-inhibitor 172 did not significantly alter GSK-mediated calcium transients in either wild-type or Trpc6 E896K/E896K platelets (Fig 8A-C). The inhibitor also had no effect on Annexin V staining regardless of genotype or stimulus ( Fig 8G). The ROCK inhibitor, Y-27632, had no appreciable effect on calcium transients in wild-type platelets, but did slightly suppress the peak Fura-2 ratio, but not AUC, in GSK-stimulated Trpc6 E896K/E896K platelets ( Fig   8D-F). Similar to a previously published report [42], Y-27632 did enhance PS exposure in wildtype platelets stimulated with thrombin ( Fig 8G). The lack of effect on thrombin-stimulated Trpc6 E896K/E896K platelets may relate to the already high degree of PS exposure under these conditions, though we cannot rule out that it is related to the modest effect of the inhibitor on the peak Fura2 ratio seen in Trpc6 E896K/E896K platelets. Finally, Y-27632 also retarded clot retraction of wild-type PRP ( Fig 8H). These results suggest a correlation between enhanced PS exposure and delayed clot retraction, whether mediated by gain-of-function TRPC6 or the ROCK inhibitor.

Discussion
By comparing the platelet phenotype of mice with wild-type, dominant negative, and gain-of-  .  P  e  n  g  F  ,  F  a  n  Y  ,  Z  h  u  X  ,  H  u  G  ,  B  u  c  h  S  J  .  T  R  P  C  c  h  a  n  n  e  l  -m  e  d  i  a  t  e  d  n  e  u  r  o  p  r  o  t  e  c  t  i  o  n  b  y  P  D  G  F  i  n  v  o  l  v  e  s  P  y  k  2  /  E  R  K  /  C  R  E  B  p  a  t  h  w  a  y  .  C  e  l  l  d  e  a  t  h  a  n  d  d  i  f  f  e  r  e  n  t  i  a  t  i  o  n  .  2  0  0  9  ;  1  6  (  1  2  )  :  1  6  8  1  -9  3  .  E  p  u  b  2  0  0  9  /  0  8  /  1  5  .  d  o  i  :  1  0  .  1  0  3  8  /  c  d  d  .  2  0  0  9  .  1  0  8  .  P  u  b  M  e  d  P  M  I  D  :  1  9  6  8  0  2  6  6  ;  P  u  b  M  e  d  C  e  n  t  r  a  l  P  M  C  I  D  :  P  M  C  2  7  8  3  9  7  6  .  3  2 .    Western blot analysis of platelet lysates from various Trpc6 mice stimulated with or without GSK as indicated. A, time-course of phospho-Erk1/2 levels in WT and KI platelets after stimulation with GSK for the indicated time. Total Erk1/2 levels were utilized as loading controls (bottom panel). B, western blot for phospho-Erk1/2 (top) and total Erk (bottom) in platelet lysates from the indicated Trpc6 genotype, treated with GSK or vehicle for 5 minutes prior to lysis. C, quantification of P-Erk1/2 to Erk ratios from western blot analysis; n=6 per group. Two-way ANOVA with Sidak's multiple comparisons test. D, western blot of KI platelets treated with or without GSK in the absence or presence of extracellular calcium. E, phospho-MLC2 (Ser19) blot of platelets unstimulated (-), or stimulated with GSK (G) or thrombin (T) for 5 minutes. Actin served as a loading control. F, quantification of phospho-MLC2 (Ser19) levels after 5 minutes of GSK stimulation; n=3. Two-way ANOVA with Sidak's multiple comparisons test.  Annexin V after being stimulated with thrombin (Thr) or thrombin plus C20; n=2 per group.
Paired t-test. seconds. Shown are mean ± SD, n=3-6. B, platelets were pretreated as in (A) followed by stimulation with ADP. Shown are mean ± SD, n=2-4. C, KI platelets were pre-incubated with (60' Fluox) or without fluoxetine followed by Fura2 analysis. Fluoxetine (1' Fluox) or vehicle (No Fluox and 60' Fluox) was added to the platelets after 20 seconds (first vertical line) followed by GSK at 80 seconds; mean ± SD, n=8. Fura-2 fluorescence ratio peak (D) and area under the cure (E) are shown as mean and individual values. One way ANOVA with Tukey's multiple comparisons test. F-G, Fura-2 imaging of KI platelets, pre-incubated with DMSO or the indicated drug (all 10 µM), and stimulated with GSK (F) or ADP (G) after 30 seconds. Shown are mean ± SD, n=5-6. H, percentage of Trpc6 E896K/E896K platelets staining for Annexin V, after pre-incubation with carrier (DMSO) or fluoxetine, followed by stimulation with vehicle (Unstim), GSK, thrombin, or thrombin plus GSK (T+G); n=6-8 animals per group. Mixed effects analysis with Sidak's multiple comparisons test.