Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice

Abstract

Background and Objective

CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region.

Methods and Results

We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLCγ2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets.

Conclusion

We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

Introduction

Platelets are the primary mediators of hemostasis and thrombosis and their activation is tightly regulated under normal physiological conditions. Upon vascular injury, circulating platelets bind to the exposed subendothelial collagen via α2β1 and signal through the glycoprotein VI (GPVI)/FcRγ-chain complex. This interaction results in the primary activation of platelets and subsequent release of the secondary mediators adenosine diphosphate (ADP) and thromboxane A₂ (TXA₂). These mediators, along with generated thrombin, further activate platelets via G protein-coupled receptor (GPCR) signaling.¹

GPVI receptors lack intrinsic receptor tyrosine kinase activity; instead, they rely on Src family kinases (SFKs) to phosphorylate the immunoreceptor tyrosine-based activation motif (ITAM) on the FcRγ chain to initiate GPVI signaling. Therefore, SFKs are indispensable for GPVI-mediated platelet activation.² Human platelets have seven SFK family members—Src, Yes, Lyn, Hck, Fyn, Fgr, and Lck, whereas mouse platelets have four—Src, Lyn, Fyn, and Fgr.² SFKs have highly conserved tyrosine residues that regulate their activation. For example, SFKs have a positive regulatory site (Y416), which is present in the activation loop between the N-terminal and C-terminal domains. The negative regulatory site (Y527) is present in the c-terminal tail.^3,4 When phosphorylated, the negative regulatory site keeps the SFKs in an inactive conformation and dephosphorylation of this residue primes SFKs for activation.⁵ Primed SFKs then undergo auto-phosphorylation at the Y416 leading to their full activation.³ Although the role of SFKs is well established in platelets, we have very little understanding of their regulation by protein tyrosine phosphatases (PTPs).

PTPs are important regulators of signal transduction.⁶ One of the most well-studied PTPs in platelets is PTP-1B, which is an essential positive regulator of Src downstream of α_IIbβ₃ activation.^7–9 Another receptor tyrosine phosphatase known to regulate SFKs is CD148. CD148-deficient platelets exhibit impaired GPVI-mediated platelet activation. However, CD148 knockout (KO) mice have a 42% reduction in GPVI platelet surface expression and thus exhibit defective platelet function.¹⁰ Thus, the mechanism behind SFK regulation in platelets remains unclear.

CD45 is a protein tyrosine phosphatase known to regulate SFKs in lymphocytes.¹¹ CD45 is a large molecule (180-220 kDa) with an extracellular, transmembrane, and catalytic cytoplasmic domain (Fig. 1). The extracellular domain consists of three different regions (1) A, B, and C regions which are encoded by exons 4, 5, and 6, respectively, and can be alternatively spliced to give various isoforms, (2) a cysteine-rich domain, and (3) a fibronectin domain.¹² All CD45 isoforms have the same cytoplasmic region, which contains domain 1 and 2. Domain 1 has phosphatase activity whereas the function of domain 2 is not known.¹³ In lymphocytes, CD45 regulates SFK activation by dephosphorylating the negative regulatory tyrosine domain.¹⁴ Given the critical role of CD45 in regulating the activation of SFKs, it would be interesting to evaluate the role of CD45 in platelets.

Fig. 1.

A representative figure showing the structure of CD45 in lymphocytes adapted from Ref. 41. CD45 consists of extracellular, transmembrane, and intracellular regions. The extracellular region of CD45 consists of alternatively spliced region liked to O-glycans, cysteine-rich region, and fibronectin domains associated with N-glycans. The intracellular region contains the D1 and D2 domain. The D1 domain is the phosphatase domain that has the catalytic activity.

Data published by Karisch et al¹⁵ in 2011 suggests that CD45 may be expressed in platelets. Using mass spectrometry, they show the presence of a c-terminal peptide of CD45. Conversely, in 1988, Terstappen and Loken¹⁶ have established that CD45 is not expressed on the surface of platelets by using flow cytometry. Based on these studies, we hypothesized that platelets express a truncated isoform of CD45, which has the catalytic cytoplasmic domain but lacks the extracellular domain. In this article, we demonstrate that platelets express a truncated CD45 isoform of approximately 65 kDa. We also show that deficiency of CD45 leads to impaired GPVI-mediated signaling and subsequent platelet function.

Experimental Procedures

Material:

Apyrase (grade VII), indomethacin, and MRS 2179 were obtained from Sigma-Aldrich (St. Louis, Missouri, United States). AR-C69931MX (cangrelor) was a gift from the Medicines Company (Parsippany, New Jersey, United States). Hexapeptides, AYPGKF, was custom synthesized at Invitrogen (Carlsbad, California, United States). Collagen-related peptide (CRP) was purchased from Dr. Richard Farndale (University of Cambridge, United Kingdom). Antibody for phosphotyrosine SFK Y416 (catalog #6943P), pSyk Y525/526 (catalog #2711), pSyk Y352 (catalog #2701), pPLCγ2 Y759 (catalog #3874), pPLCγ2 Y1217 (catalog #3871), β-Actin 13E5 (catalog #4970S), and PKC substrate antibody (catalog #22610) were purchased from Cell Signaling Technology (Danvers, Massachusetts, United States). Antibodies—CD45 H-230 (catalog #sc-25590), total Syk (Syk-01; catalog #51703), total PLCγ2 (B-10; catalog #sc-5283)—were from Santa Cruz Biotechnology (Santa Cruz, California, United States). The total pleckstrin antibody (catalog #610503) was purchased from BD Biosciences (San Jose, California, United States). Luciferin-luciferase reagent was purchased from Chrono-Log (Havertown, Pennsylvania, United States). The anti-mouse JON/A-phycoerythrin (PE), CD62-fluorescein isothiocyanate (FITC), and GPVI-FITC antibodies were obtained from Emfret Analytics (Wuerzburg, Germany). Anti-Human CD45RO PE antibody was from eBiosciences (reference #12-0457-42) and C-terminal domain-specific CD45 recombinant protein were purchased from Enzo Life Sciences (catalog #BML-SE135-0020). CD45 KO mice were obtained from Dr. Tak Mak from University Health Network in Toronto, Canada. They were bred in the central animal facility of Temple University Medical School. The CD45 exon 6 KO mice used for these studies developed subcutaneous abscesses at times. These were cleared by the veterinary staff prior to use. All other reagents were of reagent grade, and deionized water was used throughout.

Preparation of human and murine platelets:

As described in Daniel et al,¹⁷ the platelet counts for human and murine platelets was adjusted to 5 × 10⁹ cells/mL and 1.5 × 10⁸ platelets/mL, respectively.

Preparation of human platelet lysate, membrane fraction, and cytoplasmic fraction:

Note that 500 μL of Tyrode’s buffer containing 2× Halt Protease and Phosphatase cocktail solution (Pierce, Rockford, Illinois, United States) was added to 500 μLof platelets. Platelets were lysed by 4 freeze/thaw cycles and centrifuged at 1,500 × g for 10 minutes at 4°C to remove cells which are not lysed. Note that 300 μL lysed cells were mixed with 100 μL of 1% triton (whole cell lysate). Remaining part of the lysed cell suspension was ultracentrifuged at 10,000 × g for 30 minutes at 4°C. The supernatant was separated (cytoplasmic fraction). The pellet, containing the membrane and cytoskeleton, was resuspended in 100 μL of 1% TritonX-100 and spun at 12,000 × g for 10 minutes at 4°C to separate the soluble membrane fraction from the insoluble cytoskeleton. Protein estimation was performed using the Pierce BCA Protein assay kit (Thermo Scientific, Rockford, Illinois, United States). The protein concentration was adjusted to 1 μg/μL using 1 × sample buffer containing 1 mM dithiothreitol. Five or 3 μg of protein was loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel for separation and subjected to Western blotting.

Preparation of megakaryocyte lysate:

Bone marrow femurs and tibiae of wild-type (WT) mice was flushed into Iscove’s Modified Dulbecco’s Media. Megakaryocytes were separated from mononuclear cells using a discontinuous bovine serum albumin gradient (0%, 1.5%, 3%), washed in phosphate-buffered saline (PBS), and lysed using an NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, and 1:100 Halt [Thermo Fisher] protease and phosphatase cocktail). Samples were then boiled in 2× Laemmli buffer for 5 minutes prior to resolving via SDS-PAGE.

Aggregometry and Western blotting:

The aggregations, dense granule secretions, and Western blotting was performed as described in Daniel et al.¹⁷

Flow cytometry:

All data were collected using a Calibur flow cytometer (Becton Dickinson, San Jose, California, United States). Washed platelets were analyzed before and after activation with CRP and AYPGKF, and surface expression of P-selectin and JON/A binding was examined. Platelets were incubated with P-selectin-FITC or JON/A-PE antibodies and stimulated with the appropriate agonist at 37°C under non-stirring conditions. A set of unstimulated platelets was incubated with GPVI-FITC antibody. After 30 minutes of stimulation, the platelets were fixed using 1% formalin. Light scatter and fluorescence data from 10,000 platelet events were collected with all detectors in logarithmic mode. Data were analyzed using the Flow Jo software.

Pulmonary thromboembolism:

Mice were weighed, anesthetized, and injected via retro-orbital sinus with 400 μg/kg of collagen and 60 mg/kg epinephrine or PBS (control). The time to cessation of respiration was recorded. Chicago sky blue dye was perfused through the right ventricle of the heart. Lungs of the mice were examined to verify that pulmonary embolism occurred as the dye is excluded from the lungs if the circulation is obstructed due to thrombosis.

Bleeding time:

Mice (4–5 weeks old) were anesthetized using isoflurane. Four millimeters of the tail was excised and immediately immersed in 0.9% isotonic saline at 37°C. The bleeding time was defined as the time required for blood flow into the saline to stop.

Statistical analysis:

We have analyzed statistical significance of our data using unpaired t-test. Statistically significant data bearing p-value of < 0.05 are annotated by an asterisk. Data are expressed as mean ± standard error of the mean.

Results

Investigating the presence of a truncated isoform of CD45 in platelets:

First, we verified the absence of CD45 from the platelet surface using an antibody specific for the extracellular region of CD45 by flow cytometry (data not shown). Next, we investigated the presence CD45 in platelets by using an established antibody specific for the c-terminal domain of CD45¹⁸ via Western blotting. We observed that a protein of approximately 65 kDa was recognized in WT murine platelets but not in CD45 KO murine platelets (Fig. 2A). Based on the bioinformatics tools, Uniprot and ExPASy, the molecular weight of the CD45 c-terminal domain 1 and 2 is approximately 65 to 68 kDa. Thus, we believe that the observed protein is a truncated form of CD45, consisting of only a part of the c-terminal domain.

Fig. 2.

(A) Murine platelet lysate from wild-type (WT) and CD45 knockout (KO). (B) Murine megakaryocyte lysate from WT and CD45 KO mice and (C) human platelet—whole cell lysate, membrane, and cytoplasmic lysates (5 μg) were subjected to Western blot analysis using a c-terminal domain-specific antibody and β-actin was probed as a loading control. (D) CD45 c-terminal domain recombinant protein (30 and 60 ng). (E) WT and CD45 KO murine platelet lysates and (F) human platelet lysate (3 μg) was subjected to Western blotting and probed with either CD45 antibody or CD45 antibody preincubated with CD45 c-terminal domain recombinant protein. All Western blots are representative of at least three independent experiments.

Flow cytometry studies have shown the presence of CD45 on the megakaryocyte surface.¹⁹ Using Western blotting, we determined that full-length CD45 is present in WT murine megakaryocytes but not in CD45 KO megakaryocytes, confirming CD45 deletion in the megakaryocyte lineage (Fig. 2B). Additionally, we investigated the presence of CD45 in human platelets. We observed that, similar to murine platelets, human platelets also express a 65-kDa protein (Fig. 2C). In order to further evaluate the location of CD45 in human platelets, we separated the membrane and cytoplasmic fractions. We observed the 65-kDa band to be specifically present in the cytoplasmic fraction (Fig. 2C).

To validate the specificity of the CD45 antibody toward the c-terminal domain, we used a CD45 c-terminal domain recombinant protein. The molecular weight of the CD45 c-terminal domain recombinant protein is 80 kDa. The CD45 c-terminal domain recombinant protein contains amino acids from 584 to 1,281 that encompass a part of the transmembrane region and the entire c-terminal region. Therefore, the CD45 c-terminal domain recombinant protein has additional amino acids as compared to the CD45 truncated isoform expressed in platelets. The CD45 recombinant protein, WT murine platelet lysates, CD45 KO murine platelet lysates, and human platelet lysates were subjected to Western blotting and probed with either the CD45 antibody or the CD45 antibody blocked with CD45 recombinant protein. We observed that the CD45 antibody recognizes an 80-kDa recombinant protein and a 65-kDa protein in both WT murine and human platelet lysates (Fig. 2D–F). However, blocking the CD45 antibody with the CD45 recombinant protein prevents the recognition of the 80-kDa recombinant protein and a 65-kDa protein in both WT murine platelet lysates and human platelet lysates (Fig. 2D–F). These data suggest that the CD45 antibody is specific for the c-terminal domain of CD45.

Evaluation of ex vivo platelet functional responses in CD45 KO and WT mice:

We evaluated whether or not the truncated CD45 isoform has a role in platelet functional responses. Platelets have two important classes of receptors—ITAM-based receptors, such as GPVI receptors, and GPCRs, such as protease-activated receptors (PARs).¹ We stimulated WT and CD45 KO platelets with the GPVI agonists such as CRP and collagen, as well as the PAR-4 agonist AYPGKF. CD45 KO murine platelets exhibited impaired platelet aggregation and dense granule secretion when stimulated with 2 μg/mL of CRP compared to WT platelets. Upon stimulation with 10 μg/mL CRP, the differences with respect to aggregation became less apparent; however, dense granule secretion was significantly impaired in the CD45 KO murine platelets (Fig. 3A–C). Similar results were obtained using 0.5 and 1 μg/mL of the physiological GPVI agonist collagen (Fig. 3D–F). When WT and CD45 KO platelets were stimulated with 100 or 200 μM AYPGKF, there was no difference in aggregation and dense granule secretion (Fig. 3G–I). Similarly, when platelets from WT and CD45 KO mice were stimulated with low and high concentrations of 2-methylthioadenosine diphosphate, there was no observable difference in aggregation (Fig. 3J, K). These results suggest a functional role for CD45 downstream of only GPVI-mediated platelet activation.

Fig. 3.

Washed murine platelets (1.5 × 10⁸) were stimulated with low and high concentrations of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) agonists under stirring conditions. (A) Collagen-related peptide (CRP) (2 and 10 μg/mL). (B and C) Quantitation of extent of aggregation and adenosine triphosphate (ATP) secretion of (A), *p < 0.05. (D) Collagen (0.5 and 1 μg/mL). (E and F) Quantitation of extent of aggregation and ATP secretion of (D), *p < 0.05. (G)AYPGKF (100 and 200 μM). (H and I) Quantitation of extent of aggregation and ATP secretion of (G), *p < 0.05. (J) 2-Methylthioadenosine diphosphate (2-MeSADP) (100 and 30 nM). (K) Quantitation of extent of aggregation of (J). Data presented represent mean ± standard error of the mean (SEM) (n ≥ 3).

Likewise, we investigated alpha granule release and activation of α_IIbβ₃ integrin in WT and CD45 KO mouse platelets using flow cytometry. CD45 KO murine platelets showed a significant decrease in the surface expression of P-selectin (a marker for α-granule release) as compared to WT murine platelets when stimulated with CRP (2 and 10 μg/mL) (Fig. 4A). Furthermore, we investigated the activation of α_IIbβ₃ using JON/A antibody, which binds only to active α_IIbβ₃. We observed decreased levels of JON/A binding in CD45 KO murine platelets stimulated with CRP (2 and 10 μg/mL) as compared to WT (Fig. 4B). We did not observe any differences in alpha granule secretion or activation of α_IIbβ₃ between WT and CD45 KO platelets when stimulated with AYPGKF (data not shown).

Fig. 4.

(A, B) Washed murine platelets were stimulated with agonist for 30 minutes at 37°C in the presence of fluorescein isothiocyanate (FITC)-labeled anti-mouse CD62 and phycoerythrin (PE)-labeled anti-mouse JON/A antibodies (Becton Dickinson, San Jose, California, United States). CD62 and JON/A positive platelets were gated and analyzed for P-selectin expression and αIIbβ3 activity, respectively. The difference in mean fluorescence intensity (MFI) of CD62 and JON/A binding between knockout (KO) and wild-type (WT) murine platelets was calculated. Data are expressed as percent over the basal signal (n = 4), *p < 0.05. (C) Washed WT and CD45 KO murine resting platelets were incubated with a FITC-labeled anti-mouse glycoprotein VI (GPVI) antibody. The gray and black histograms represent the WT and CD45 KO surface expression, respectively, whereas the light gray histogram represents the immunoglobulin G (IgG) control, data are mean ± standard error of the mean (SEM) (n = 5), *p < 0.05. (D) Quantitation of GPVI surface expression on WT and CD45 KO mouse platelets.

It was previously revealed that the absence of CD148 phosphatase results in a 42% reduction of GPVI surface expression on the platelet surface.¹⁰ A decrease in the surface expression of GPVI would explain why the authors saw impaired GPVI-mediated platelet functional responses. Hence, we evaluated the GPVI surface expression on CD45 KO and WT platelets using flow cytometry. We observed that the CD45 KO and WT mouse platelets had similar GPVI surface expression (Fig. 4C). This indicates that CD45 does not regulate GPVI surface expression in platelets and cannot explain the reduction of CRP-induced platelet functional responses in the CD45 KO murine platelets.

Evaluation of in vivo platelet functional responses in CD45 KO and WT mice:

As CD45 KO platelets exhibited impaired GPVI-mediated ex vivo platelet functional responses, we investigated the physiological implications of CD45 deficiency. Initially, we measured tail bleeding time, which reflects primary hemostatic function. We observed that some CD45 KO mice had prolonged tail bleeding and some had similar bleeding times as the WT mice (Fig. 5A). Therefore, the average bleeding time of CD45 KO mice is higher as compared to the WTs.

Fig. 5.

(A) Bleeding time was measured in wild-type (WT) and CD45 knockout (KO) mice following excision of the distal 4 mm of the tail. Data are mean ± standard error of the mean (SEM) (CD45 KO n = 26, WT n = 32), *p < 0.05. (B) WT and CD45 KO mice were anesthetized and injected either with phosphate-buffered saline (PBS) or appropriate amounts of collagen and epinephrine and time to cessation of respiration was recorded. Data are mean ± SEM (n = 4), *p < 0.05.

We also used a model of pulmonary thromboembolism to evaluate the ability of platelets to form a thrombus in vivo.^20–22 We injected 400 μg/kg of collagen and 60 mg/kg of epinephrine into the retro-orbital space of WT and CD45 KO mice. This leads to activation of platelets in circulation and formation of emboli, which travel to the lungs and block blood flow thereby resulting in cessation of respiration. We observed that the time to cessation of respiration was prolonged in CD45 KO as compared to WT, which is indicative of a defect in thrombus formation (Fig. 5B). We confirmed that mortality was due to pulmonary embolism by injecting Chicago sky blue dye in the right ventricle of the heart. This dye is excluded from the lungs if the circulation is obstructed due to thrombosis (data not shown). Thus, we concluded that CD45 KO mice have impaired thrombosis and hemostasis.

Evaluation of GPVI signaling in CD45 KO and WT murine platelets:

Since CD45 KO murine platelets were defective in GPVI-mediated platelet functional responses, we evaluated the signaling downstream of the GPVI receptor. First, we investigated the phosphorylation status of two proximal signaling molecules in the GPVI pathway—Syk and PLCγ2. When CD45 KO murine platelets were stimulated with 2 μg/mL CRP (Supplementary Fig. S1, available in the online version) or 10μg/mL CRP(Fig. 6A–F) for 10 and 30 seconds, we saw a significant decrease in the phosphorylation of Syk (Y352, Y525/526) and PLCγ2 (Y759, Y1217) compared to WT control platelets. Consistently, using a low concentration of CRP (1 μg/mL), we observed reduced pleckstrin phosphorylation in CD45 KO platelets (Fig. 6G, H), suggesting that subsequent calcium release may be impaired.

Fig. 6.

(A) Murine platelets from wild-type (WT) and CD45 knockout (KO) mice were stimulated with the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP) (10 μg/mL) for 10 and 30 seconds and reaction was stopped using 1/10 volume 6.6 N HClO₄. Samples were centrifuged and the pellets were washed and dissolved in sample buffer. These lysates were subjected to Western blot analysis and probed with pSyk Y525/ 526, pSyk Y352, pPLCγ2 Y759, and pPLCγ2 Y1217 antibodies. The same blot was probed with total Syk and total PLCγ2 antibodies as protein loading control in each lane. (B-F) Quantitation for phosphorylation of Syk and PLCγ2, data are mean ± standard error of the mean (SEM) (n = 4), *p < 0.05. (G) Washed platelets from WT and CD45 KO mice were stimulated or not with CRP (1 mg/mL) for 60 seconds at 37°C with stirring. The reaction was stopped with 1/10 volume 6.6N HClO₄. The resultant precipitates were washed and resuspended with sample buffer prior to separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following transfer to nitrocellulose, the membranes were probed for PKC substrate and total pleckstrin. (H) Quantification from (A) expressed as mean ± SEM of PKC substrate phosphorylation/pleckstrin (n = 3). *p < 0.05.

SFKs are upstream of Syk and are indispensable for GPVI receptor activation.² CD45 is known to regulate SFK activation in lymphocytes by dephosphorylating the negative regulatory tyrosine.¹⁴ Thus, we evaluated whether or not the truncated isoform of CD45 regulates SFK phosphorylation in platelets. However, SFKs are also important for signaling downstream of GPCRs.^2,23 GPCRs (P2Y12, P2Y1, and TP receptor) are stimulated via secreted TXA₂ and ADP, which act in an autocrine and paracrine fashion leading to further activation of platelets.²⁴

To evaluate SFK phosphorylation downstream of only the GPVI receptor, we preincubated the platelets with the feedback inhibitors indomethacin, which inhibits TXA₂ generation, and the ADP receptor antagonists, MRS-2179 and ARC-69931 (cangrelor). This was followed by stimulation of the platelets with 10 μg/mL of CRP. Using the feedback inhibitors enabled us to eliminate any contribution by TXA₂ and ADP to SFK phosphorylation. Under these conditions, we observed that CD45 KO murine platelets have reduced CRP-induced SFK Y416 phosphorylation compared to WT murine platelets (Fig. 7). Therefore, we concluded that the truncated isoform of CD45 regulates SFK phosphorylation in platelets.

Fig. 7.

(A) Platelets from wild-type (WT) and CD45 knockout (KO) mice were incubated with secondary signaling inhibitors—indomethacin 10 μM, MRS-2179 100 μM, ARC-69931 (cangrelor) 100 nM for 5 minutes—and then stimulated with glycoprotein VI (GPVI) agonist collagen-related peptide (CRP) (10 μg/mL) for 10 and 30 seconds and reaction was stopped using the 1/10 volume 6.6 N HClO₄. Samples were centrifuged and the pellets were washed and dissolved in sample buffer. These lysates were subjected to Western blot analysis using pSFK Y416 antibody. The same blot was probed with total PLCγ2 antibody as protein loading control in each lane. (B) Quantitation for phosphorylation of Src family kinase (SFK), data are mean ± standard error of the mean (SEM) (n = 3), *p < 0.05.

Discussion

Numerous isoforms of CD45 exist in different cells. Lymphocytes express various isoforms on their surface depending upon their stage of development. The majority of developing thymocytes and activated T cells express a low molecular weight isoform of CD45—RO—whereas the mature T cells express multiple isoforms. The human Langerhans cells express an unusual isoform that is similar to the RO (180 kDa) epitope but lacks the oligosaccharide with terminal sialic acid moiety.²⁵ Also, a report from Kirchberger et al²⁶ demonstrated the presence of another uncommon CD45 isoform in monocytes and neutrophil granulocytes. They found that upon stimulation of phagocytes, a 95-kDa fraction of CD45 consisting of the cytoplasmic tail is cleaved by proteases. This 95-kDa fraction of CD45 aligns with the c-terminal region of CD45 and is cytoplasmic.²⁶

Our study shows that platelets express a truncated isoform of CD45 of approximately 65 kDa, and consists of only a part of the c-terminal region. It is important to note that the c-terminal region of CD45 contains a catalytic domain which is indispensable for its function.²⁷ Since the extracellular domain of CD45 in lymphocytes does not contribute to the catalytic activity of CD45,¹¹ it is likely that the truncated isoform of CD45 functions even in the absence of the extracellular domain. Furthermore, the data sheet of the recombinant CD45 c-terminal domain protein used in Fig. 2 suggests that it is a catalytically active protein. This further supports our above hypothesis.

We also investigated the presence of CD45 in megakaryocytes. Flow cytometry studies have shown that megakaryocytes express a full-length isoform of CD45.¹⁹ We have verified this via Western blotting. We observed that the CD45 antibody recognized a 180-kDa protein in WT but not in the CD45 KO. Also, the 65-kDa protein observed in platelets was not present in the megakaryocytic protein lysates. Therefore, we ruled out the possibility that the truncated CD45 is an alternatively spliced variant. Previous studies in phagocytes have demonstrated that a fraction of CD45 is cleaved sequentially by serine/metalloproteases and γ-secretase upon stimulation for activation by fungal stimulation.²⁶ We speculate that the truncated CD45 isoform is generated by proteolytic cleavage when platelets are formed from megakaryocytes by a mechanism that involves one or several proteases. Currently, we do not know the exact residue where full-length CD45 is cleaved and this needs to be investigated in the future. Our data also suggest that human platelets express a 65-kDa of CD45, which is present in the cytosol. A 100-kDa band is recognized by the antibody only in the membrane fraction and not in other lanes. We predict that the 100-kDa band is a nonspecific band and is recognized due to the enrichment of membrane bound proteins in the membrane fraction. The whole cell lysate would contain both membrane and cytosolic proteins, which would serve to dilute the membrane fraction. Therefore, we do not see a prominent 100 kDa band in the whole cell lysate.

CD45 plays a very crucial role in all hematopoietic cells studied^28–33 however, the function of CD45 in platelets had never been evaluated. In this article, we demonstrate that the truncated isoform of CD45 is a positive regulator of SFK activation downstream of the GPVI receptor. Consequently, the absence of CD45 specifically impairs GPVI-mediated platelet signaling and platelet functional responses. However, CD45 deficiency does not completely abolish the platelet signaling or functional response, which suggests that other phosphatases might play a role in GPVI signaling. We speculate that another phosphatase, CD148, plays a role in regulating SFK activation along with CD45. Although the CD148 KO studies suggest that the impaired GPVI responses are due to the decreased GPVI surface expression, the in vitro studies using recombinant CD148 have shown that CD148 recombinant protein directly dephosphorylates the c-terminal inhibitory and activation loop sites of SFKs.¹⁰ Therefore, CD45 and CD148 both may play a role in regulating the SFK activation in platelets.

Stimulation of CD45 KO murine platelets with lower concentrations of GPVI agonist CRP results in impaired platelet aggregation and secretion in CD45 KO murine platelets. With an increase in the CRP concentration, the WT and CD45 KO platelets show the same extent of aggregation. However, the dense granule secretion is impaired at both low and high concentrations of CRP. This discrepancy in aggregation and secretion at higher concentration of CRP is explained by the fact that aggregation saturates at a relatively low concentration of the agonist.

We also evaluated the alpha granule secretion and α_IIbβ₃ integrin activation in the CD45 KO platelets stimulated with CRP. CD45 KO platelets show a defect in alpha granule secretion and α_IIbβ₃ integrin activation at both low and high concentrations of CRP. It is interesting that at higher concentrations of CRP, although α_IIbβ₃ integrin activation is impaired, the CD45 KO murine platelets aggregate fully. However, we can explain this observation using the example of patients who are heterozygous for Glanzmann’s thrombasthenia. These patients have a 50% reduction in α_IIbβ₃ integrin activation; however, there are no demonstrable defects in aggregation.^34,35 This demonstrates that 50% α_IIbβ₃ integrin activation is sufficient for full aggregation.

Because of the defects noted in the response of CD45 KO platelets to the GPVI agonist CRP, we investigated GPVI signaling in CD45 KO murine platelets. SFKs are essential for signaling downstream of GPVI receptors. SFK activation is dependent on dephosphorylation of the negative regulatory site Y527 that leads to a partially active conformation of SFKs. SFKs then undergoes auto-phosphorylation at the Y416 residue to attain fully active conformation. CD45 KO murine platelets stimulated with GPVI agonist in presence of feedback inhibitors show reduced SFK Y416 phosphorylation as compared to the WT platelets. This indicates that absence of CD45 results in impaired SFK activation downstream of the GPVI receptor. We speculate that the truncated CD45 dephosphorylates the negative regulatory tyrosine residue and activates SFKs. Therefore, in the absence of CD45, there is no dephosphorylation of the negative regulatory tyrosine residue and thus no further activation of SFKs.

Syk and PLCγ2 phosphorylations are dependent on SFK activation. Activated SFKs phosphorylate the tyrosine residues on the ITAM domain of the FcRγ chain, thereby creating docking sites for Syk recruitment and activation.³⁶ Syk undergoes auto-phosphorylation on tyrosine residues including Y352 and Y525/526. Activation of Syk is followed by downstream signaling which eventually leads to phosphorylation of PLCγ2.³⁷ Therefore, our observation that SFK Y416 phosphorylation is reduced in CD45 KO mice compared to WT following stimulation with a GPVI agonist is supported by reduced Syk and PLCγ2 phosphorylation also observed in CD45 KO platelets.

Furthermore, we evaluated the physiological implications of CD45 deficiency by measuring hemostasis via tail bleeding. Although the average bleeding time of CD45 KO mice was higher compared to WT mice, it is clear that the mice formed two distinct groups. One group bled for a very short time period, while the other group bled for a longer time period. A similar trend has been observed in GPVI KO mice as well as CD148 KO mice.^38,39 Bleeding time of GPVI KO mice is controversial. One study has shown that there is no prolongation of bleeding times in mice with GPVI deficiency.⁴⁰ In another study, the authors reported bleeding times in 262 GPVI KO mice out of which 120 GPVI KO mice show prolonged bleeding times and 66 GPVI KO mice showed bleeding times similar to the WT. This group has shown that the variability in bleeding times in GPVI KO mice is due to the Modifier of hemostasis (Mh) locus on chromosome 4, which controls in vivo hemostasis of GPVI KO mice. In the future it would be interesting to evaluate whether or not the variability in bleeding times for CD45 KO mice is due to the Mh locus.

Next, we evaluated in vivo thrombosis in CD45 KO mice using a model of pulmonary embolism. We found that the time to cessation of respiration was higher in CD45 KO mice compared to WT mice, which is indicative of the fact that in vivo platelet activation in CD45 KO mice is reduced compared to WT platelet activation. To induce platelet activation, we injected collagen and epinephrine intravenously. Our in vitro data suggest that CD45 KO platelets have defective GPVI (receptor for collagen) signaling. These in vivo data support our assertion that CD45 is a positive regulator of thrombosis.

In conclusion, we show that CD45 is expressed in platelets as a truncated isoform that regulates GPVI-mediated platelet functional responses through SFK activation.

What is known about this topic?

• CD45 is a protein tyrosine phosphatase that regulates SFKs in lymphocytes.

• CD45 has an intracellular region that contains a phosphatase domain.

• The presence of CD45 in platelets has not been established.

What does this paper add?

• A truncated isoform of CD45 is present in platelets.

• CD45 positively regulates SFK activity downstream of GPVI.

• Thrombosis and hemostasis are impaired in CD45 knockout mice.