Mechanism of race-dependent platelet activation through the PAR4 and Gq signaling axis

Abstract

Objective

Black individuals are at an increased risk of myocardial infarction and stroke, two vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to anti-platelet therapies.

Approach and Results

Washed human platelets from black volunteers were hyper-aggregable in response to PAR4-mediated platelet stimulation compared to whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP, suggesting that the racial difference is independent of secondary feedback. Further, stimulation of platelets from black donors with PAR4-activating peptide (PAR4-AP) showed a potentiated level of activation through the G_q pathway compared to platelets from white donors. Differences in signaling included increased Ca²⁺ mobilization, Rap1 activation, and integrin αIIbβ3 activation with no observed difference in platelet protein expression between the groups tested.

Conclusions

Our study is the first to demonstrate that the G_q pathway is differentially regulated by race following PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of COX and P2Y₁₂ dual inhibition, suggesting that current anti-platelet therapy may provide less protection to blacks than whites.

Introduction

Platelet activation is a key step in the formation of an occlusive thrombus. Thrombotic events precipitated by vascular disease can lead to stroke or myocardial infarction (MI), two of the leading causes of death in the United States.^{1, 2} Further, MI and stroke disproportionally burden black individuals relative to white individuals.^3–6 Due to the importance of thrombosis in the pathogenesis of MI and stroke, the activation of platelets from healthy black or white donors in response to a panel of common platelet agonists was recently tested by our group.⁷ Interestingly, signaling through one of the two thrombin receptors expressed on the human platelet, protease-activated receptor-4 (PAR4), resulted in greater aggregation in platelets from black volunteers as compared to white volunteers.

Thrombin is a potent platelet agonist whose regulation of platelet function has been extensively studied.^8–10 Thrombin initiates platelet activation through the enzymatic cleavage of the N-terminal extracellular domain of the PARs. This new N-terminus serves as a tethered ligand that activates PAR G protein-coupled receptor signaling.¹¹ Human platelets express two PARs, PAR1 and PAR4, that signal through both overlapping as well as unique biochemical steps.^12–15 Specifically, the stimulation of PARs leads to the activation of the G_12/13 and G_q pathways.¹⁶ The initiation of G_q signaling leads to a rise in intracellular Ca²⁺, which activates Rap1, culminating in the activation of αIIbβ3, a surface receptor required for platelet aggregation.¹⁷ While this pathway has been extensively studied, the component(s) that may be differentially regulated by race remain unclear. The current study sought to determine how PAR4 reactivity is differentially regulated between healthy black and white donors.

In the presence of PAR1 antagonist, platelets from black donors are hyper-responsive to thrombin compared to platelets from white donors.⁷ As PAR1 antagonists have been approved for use in the clinic, understanding the differential signaling elicited by thrombin activation through PAR4 will have important clinical implications. In this study we show that racial differences in the PAR4 G_q signaling pathway including Ca²⁺ mobilization, Rap1 activation, and αIIbβ3 activation contribute to the racial difference observed in PAR4-mediated platelet aggregation. Understanding these differences that occur in the PAR4 pathway in platelets may lead to targeted therapies to reduce thrombotic risk in black individuals.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

PAR4-mediated aggregation is differentially regulated by race

Previously published work suggests a racial component exists for PAR4-mediated platelet aggregation.⁷ While that study was conducted on over 150 healthy individuals, recruitment was primarily limited to a localized region (Houston, TX). To determine if differences in PAR4-AP-mediated platelet aggregation are due to racial differences and not unique to the southwestern United States, PAR4-AP platelet aggregation was measured in platelets from healthy black and white subjects from the Philadelphia region. Platelets from black donors in platelet rich plasma (PRP) stimulated with PAR4-AP had an increase in maximum aggregation relative to platelets in PRP from white donors with significant differences in maximal aggregation between black and white donors being observed at 50µM and 75µM PAR4-AP (Fig 1A-B). No difference in PAR4-AP-induced aggregation was observed at higher concentrations of PAR4-AP (100 µM). In addition to platelets, PRP contains other plasma constituents which may be differentially expressed by race and indirectly influence platelet activity. No difference in PAR4-mediated platelet aggregation was observed when washed platelets from white donors were resuspended in platelet poor plasma (PPP) from black donors or vice versa. (Fig 1C+D) This data suggest that the components of plasma are not a major contributor to the racial difference in PAR4-mediated platelet aggregation.

Figure 1. Platelets from blacks are hyper-aggregable to PAR4-AP stimulation, as compared to whites.

(A) Representative aggregation tracings of platelets in PRP from a white or black donor in response to 75 µM PAR4-AP. (B) Quantification of max aggregation of black (n=8) or white (n=8) platelets in PRP in response to an increasing concentration of PAR4-AP. (C) Washed platelets from white donors were resuspended in their own platelet poor plasma (PPP) or PPP from a black donor than stimulated with increasing concentrations of PAR4-AP. (D) The reciprocal experiment from panel C was performed with washed platelets from black donors. Representative aggregation tracings of washed platelets from black and white donors in response to 25 µM (E) or 50 µM (F) of PAR4-AP. The aggregation of washed platelets from black and white donors in response to increasing concentrations of PAR4-AP was evaluated by both maximum aggregation (G) (n=14–22) and time to 50% aggregation (H) (n=5–10). Data represents mean ± S.E.M. Data was analyzed using a two-way ANOVA.

To eliminate potential confounding factors in the plasma, platelets were isolated from the plasma by centrifugation and the activation of washed platelets was measured in response to stimulation with thrombin, PAR1-AP or PAR4-AP. Consistent with platelet responses observed in PRP (Fig 1A-B), the maximal aggregation of washed platelets from black and white donors stimulated with thrombin (Supplemental Fig IA) or PAR1-AP (Supplemental Fig IB) was the same, but enhanced in PAR4-AP stimulated platelets from black donors compared to white donors with significant differences in aggregation by race being observed with as little as 10 µM PAR4-AP (Fig 1E-H). Maximal platelet aggregation and time to 50% aggregation in response to PAR4-AP were evaluated in platelets from blacks (Fig 1G and 1H, respectively), both of which demonstrated that platelets from black donors were hyper-aggregable compared to those from white donors in response to PAR4-AP. Washed platelets were observed to be more sensitive to PAR4-AP stimulation compared to platelets in PRP, possibly due to nonspecific binding of the agonist peptide to other plasma proteins. To determine if potential differences exist in the kinetics of platelet activation, the time to 50% aggregation was measured, which encompasses both the lag time (the time from agonist addition until the start of aggregation) as well as the rate of aggregation. Since not all donors aggregated in response to threshold doses of PAR4-AP, platelets from subjects that failed to reach 50% aggregation were eliminated from analysis. For the donors that did not reach 50% aggregation, 6 of 13 white donors at 35 µM of PAR4-AP and 3 of 12 white donors at 50 µM of PAR4-AP were excluded from analysis. Even with the absence of a number of white subjects due to lack of platelet aggregation, platelets from white donors were slower to 50% aggregation compared to platelets from black donors at low concentrations of PAR4-AP (35–50 µM) (Fig 1H). These data suggest that the racial difference in PAR4-mediated platelet activation is intrinsic to the platelet.

Racial differences in PAR4-mediated platelet activation are independent of COX and P2Y₁₂

Platelet aggregation occurs in two phases in response to weak stimulation such as low concentrations of PAR4-AP. Weak agonists induce the primary phase of aggregation, which then incites the generation or release of soluble platelet agonists such as adenosine diphosphate (ADP) and arachidonic acid (AA), which are capable of eliciting a secondary phase of aggregation.^{14, 18, 19} While no racial differences in platelet aggregation were found with ADP and AA, a precursor to thromboxane (TxA₂), these tests were performed with only a single concentration of each agonist. It is therefore possible that racial differences occur at other concentrations of ADP and AA which were not tested in previous studies.⁷ To determine if racial differences in PAR4-AP-mediated platelet aggregation were due to differences in secondary feedback signaling through ADP or TxA₂, platelets were stimulated with PAR4-AP in the presence of aspirin, a COX inhibitor that blocks TxA₂ production, and 2MeSAMP, a P2Y₁₂ (ADP receptor) antagonist. Platelets from blacks remained hyper-aggregable to PAR4-AP stimulation compared to whites when treated ex vivo with either 2MeSAMP (Fig 2A), aspirin (Fig 2B), or both (Fig 2C + Supplemental Fig II). While dual inhibition appeared to have a synergistic inhibitory effect on platelet aggregation, the persistence of racial differences in platelets stimulated with PAR4-AP in the presence of dual platelet inhibitors suggests that these differences are at least partially independent of TxA₂ and ADP signaling. Additionally, to assess if there was a racial difference in TxA₂ receptor-induced platelet aggregation, washed platelets from black or white donors were stimulated with increasing concentrations of the thromboxane mimetic, U46619. There was no racial difference in maximal platelet aggregation in response to U46619 (Supplemental Fig IC).

Figure 2. The racial difference in PAR4 mediated platelet activation persists in the presence of ex vivo COX and P2Y₁₂ inhibition.

Washed platelets from black (n=8) or white (n=8) donors were pretreated with either 2MeSAMP (A) for 20 minutes, aspirin (B) for 40 minutes, or both (C) prior to PAR4-AP stimulation. Each figure contains a representative tracing of platelets from a black and white donor at the concentration where the racial difference is maximized. Data represents mean ± S.E.M. Data was analyzed using a two-way ANOVA.

PAR4 surface expression

Platelets express appreciable amounts of PAR1 and PAR4 on their surface.¹⁰ One possible explanation for the racial difference in PAR4-mediated platelet aggregation is an increase in the surface expression of PAR4 on platelets from black donors relative to platelets from white donors. To determine if this was the case, surface expression of PAR4 was measured in platelets from white and black donors. Resting platelets were incubated with a FITC-conjugated PAR4 antibody to measure the amount of PAR4 expressed on the surface of platelets. No significant difference was observed between the two groups. (Fig 3A-B).

Figure 3. PAR4 surface expression is same between platelets from blacks and whites.

PAR4 surface expression of resting platelets from blacks and whites was measured by flow cytometry. (A) A representative histogram of the PAR4 surface expression on platelets from a black or white donor. (B) Graphs depicting the PAR4 surface expression of Blacks (n=5) and Whites (n=6). Data represents mean ± S.E.M. (one-tailed t-test)

Ca²⁺ mobilization is differentially regulated by race through PAR4

Since no racial difference was observed in the surface expression of PAR4, the activation status of key biochemical components of the PAR4 pathway was assessed in platelets from blacks and whites. PAR4 stimulation of platelets leads to a rise in intracellular Ca²⁺, which is critical for some of the key steps leading to normal PAR4-mediated platelet aggregation including protein kinase C (PKC), Rap1, and granule secretion.^{12, 20} Platelets from black donors stimulated with PAR4-AP (50 µM) had a significantly higher increase in maximal Ca²⁺ mobilization compared to platelets from white donors (Fig 4A-B).

Figure 4. Ca²⁺ mobilization is elevated in PAR4-AP stimulated platelets from Blacks compared Whites.

(A) Representative tracing of Ca²⁺ mobilization in platelets from a black and white donor stimulated with 50 µM PAR4-AP. Data is normalized to baseline MFI prior to stimulation (B) Graph depicting maximum Ca²⁺ mobilization in platelets stimulated with 50 µM PAR4-AP from white (n=11) and black (n=8) donors. Data represents mean ± S.E.M. (* P<0.05, one-tailed t-test)

Rap1 and PKC activity are differentially regulated by race

Rap1 and PKC are regulated in part by Ca²⁺ mobilization. Since Ca²⁺ mobilization was potentiated in platelets from black donors (Fig 4), it was important to identify if the difference in free Ca²⁺ in the platelet following PAR4-AP translates to a difference in activation of these key biochemical intermediates in platelet aggregation. Rap1, a small G-protein, is known to be important for inside-out activation of α_IIbβ₃, an essential step in platelet aggregation.²¹ Rap1 is activated as part of the PAR4 G_q pathway downstream of Ca²⁺ mobilization.²² To determine if the racial difference in Ca²⁺ mobilization leads to a difference in Rap1 activation between platelets from white or black donors stimulated with PAR4-AP, Rap1 activity was measured over time following stimulation. Rap1 activation was elevated in platelets from black donors at each time point measured and was significantly higher in platelets from black donors at 5 minutes following stimulation with 50 µM PAR4-AP (Fig 5A-B).

Figure 5. The activation of Rap1 and PKC are enhanced in platelets from Blacks upon PAR4 stimulation compared to Whites.

The activation of Rap1 and PKC was measured in platelets from black or white donors stimulated with 50 µM PAR4-AP for an increasing amount of time. (A) Representative Western blots of the active and total amount of Rap1 in platelets from white or black donors. (B) Quantification of Rap1 activation in PAR4 stimulated platelets from Blacks (black bars, n=8) and Whites (white bars, n=8). (C) Representative Western blots of the amount of Pleckstrin phosphorylation by PKC and the total Pleckstrin in each sample. (D) Graph depicts the amount of Pleckstrin phosphorylation normalized to the amount of total Pleckstrin in each sample in platelets from Blacks (n=7) and Whites (n=7). Data represents mean ± S.E.M (* P<0.05, one-tailed t-test)

PKC activation is a key intermediate step downstream of PAR4 G_q signaling in the platelet. Ca²⁺ is known to be essential for the activation of several PKC isoforms involved in platelet function.²³ Therefore, to determine if PKC was differentially regulated by race, PKC activation was measured by its ability to phosphorylate Pleckstrin, a major PKC substrate in platelets, following stimulation by PAR4-AP. Following stimulation with PAR4-AP, platelets from black donors showed a significantly potentiated phosphorylation of Pleckstrin compared to platelets from whites at 30 seconds while no difference in total Pleckstrin levels was observed between donors (Fig 5C-D). Taken together, a measurable difference was observed in the level of platelet function through the PAR4 pathway in platelets from black and white donors. Specifically, the data suggests that a racial difference in platelet Ca²⁺ mobilization and its downstream effectors, Rap1 and PKC play at least a partial role in mediating these differential effects (Figs 4–5).

Integrin αIIbβ3 differentially regulated through PAR4

Since Rap1 is known to be an essential regulator of integrin αIIbβ3 activation²¹ and its activity is differentially regulated by race (Fig 5), it is likely that αIIbβ3 activity is also potentiated in platelets from blacks stimulated with PAR4-AP compared to whites. To identify if the potentiated activity observed in platelets from black donors is measurable at the level of αIIbβ3, platelets from black and white donors were stimulated with PAR4-AP in the presence of FITC-conjugated PAC-1, an antibody specific for the active conformation of αIIbβ3 and the level of active αIIbβ3 was measured on the surface of platelets by flow cytometry. Upon stimulation with 50 µM of PAR4-AP, platelets from black donors had significantly increased levels of active αIIbβ3 compared to platelets from white donors (Fig 6A-B).

Figure 6. αIIbβ3 activation in platelets from Blacks relative to Whites.

The activation status of αIIbβ3 in washed platelets from white and black donors stimulated with 50 µM PAR4-AP was measured by flow cytometry. A) Representative histograms of platelets from white and black donors stimulated with PAR4-AP for 300 seconds. (B) Graph representing the MFI of platelets from black (n=15) or white (n=16) donors stimulated with PAR4-AP at various times. The protein expression of αIIb, Rap1, and GAPDH was measured in resting platelets from black (n=8) and white (n=10) donors by Western blot. (C) Representative blot of αIIb, Rap1, and GAPDH. Graphs depicting the levels of (D) αIIb or (E) Rap1 normalized to the amount of GAPDH. Data represents mean ± S.E.M. (*P<0.05, one-tailed t-test)

Potentiation of the biochemical intermediate steps in PAR4 signaling in the platelet in blacks may be due to a number of factors including altered receptor number, variability of the signalosome, or genetic variability in expression levels of the proteins involved in the signaling following PAR4 activation. To determine if the increased signaling in platelets from black donors was due to a differential expression of some of the key signaling proteins in the PAR4 pathway, protein levels for Rap1 and αIIbβ3 were measured from resting platelets from black and white donors. Surprisingly, all donors were observed to have similar levels of these proteins as measured by Western blot and normalized to GAPDH (Fig 6C-E). This data suggests potential differences in the expression of these key intermediates is not the determining factor mediating differential platelet activity by race through the PAR4 pathway.

Discussion

Thrombotic diseases such as MI and stroke disproportionally burden blacks.^3–6 Therefore, our group sought to investigate the underlying biochemical differences mediating the observed racial difference in platelet reactivity. Recently, we were able to show that platelets from black donors were hyper-aggregable to PAR4 stimulation compared to platelets from white donors.⁷ However, the mechanism responsible for this difference in signaling remained unclear. We demonstrate here for the first time that the racial difference in PAR4-mediated platelet aggregation is due to potentiation of signaling components in the PAR4 G_q pathway of platelets from blacks compared to whites. The racial difference in PAR4-mediated platelet activation was not due to differences in the protein expression of key PAR4 signaling components including the receptor (Fig 3), the small G protein Rap1, and the integrin αIIbβ3 (Fig 6). The observation that Rap1 and αIIbβ3 are not differentially expressed by race is consistent with previous findings that platelets from blacks and whites have similar levels of aggregation to all other agonists tested except PAR4-AP.⁷ The magnitude due to race in the difference of activation of the PAR4 G_q pathway components tested including Ca²⁺ mobilization (Fig 4), Rap1(Fig 5) and αIIbβ3 (Fig 6) is similar with roughly a 1.5 to 2 fold difference. This data is supportive of racial differences in PAR4-mediated platelet activation being propagated through differences in Ca²⁺ mobilization and signaling components upstream of Ca²⁺ that may contribute to differences in platelet function.

Our previous study, which first identified a racial difference in platelet aggregation, consisted of a cohort recruited primarily from the Houston area (with a small cohort from the Philadelphia area used in thrombin studies). It is possible that the racial difference observed in PAR4-mediated platelet activation could be due to unique genetic or environmental factors in blacks and/or whites in that geographic region. The present study therefore sought to determine if hyper-aggregation in platelets from blacks in PRP compared to whites was genetic or a combination of genetic and environmental. The current findings are supportive, based on the newly recruited cohort from Philadelphia that the difference in PAR4-mediated platelet aggregation is due to racial rather than regional differences. Additionally, while aggregation studies using PRP are more physiologically relevant than washed platelets, the components of the plasma including lipids and proteins can confound the interpretation of potential differences inherent in platelet signaling from these groups. Hence, aggregation studies were performed with isolated washed platelets supporting that the observed racial difference in platelet aggregation are due to an inherent difference in the platelets themselves (Fig 1G-H).

The racial difference in PAR4-mediated platelet aggregation in washed platelets occurred at sub-maximal concentrations of PAR4-AP (less than 60 µM) (Fig 1G). While the entire dose-response for aggregation to PAR4-AP was substantially shifted to the left in platelets from black donors compared to those from white donors, the largest differences in aggregation were observed in the dynamic range for PAR4-AP-mediated aggregation occurring below the EC₈₀. As many studies have focused on high concentrations of PAR4-AP (greater than EC₁₀₀), it is possible that potential racial differences in these platelet function studies were overlooked. Further, at lower concentrations of PAR4-AP secondary agonists generated or released by platelets play a more important role in mediating full platelet aggregation.^{14, 18, 19, 24}

We demonstrate that a racial differences in PAR4-mediated aggregation still occur even in the presence of a COX inhibitor and P2Y₁₂ antagonist, which block the production of TxA₂ from AA and ADP signaling respectively (Fig 2).Together this data suggests the hyper-activation of PAR4 stimulated platelets from blacks compared to those from whites is due in part to differences in the expression or activation of an effector within the PAR4 pathway. The current study identified a number of biochemical intermediate steps in the PAR4 pathway which are differentially regulated by race from mobilization of intracellular Ca²⁺ to αIIbβ3 activation and aggregation. The role of Ca²⁺ in differential signaling in the platelet by race is consistent with previous studies that reported a racial difference in Ca²⁺ mobilization in platelets.²⁵ Interestingly, previous studies have also shown the kinetics of Ca²⁺ mobilization to be dependent on a number of factors including the method of activation. For example, PAR1 and PAR4 stimulated platelets show differing kinetics for Ca²⁺ mobilization,¹⁵ suggesting that Ca²⁺ release is uniquely regulated in these two pathways.¹² Additionally, studies have shown in other cell types that Ca²⁺ mobilization is differentially regulated by race due to differences in ion channels such as sodium and potassium transporters.²⁶ Within platelets, Ca²⁺ release is regulated by a complex signaling network, including IP₃R, STIM1, Orai1, and TRPC6, all of which may be involved in the differential signaling observed in Ca²⁺ mobilization following stimulation of the PAR4 pathway in blacks and whites.²⁷ Future work is required to determine if the components responsible for the regulation of Ca²⁺ mobilization contribute to the racial difference in PAR4-mediated platelet activation. Another possibility underlying the potentiation in Ca²⁺ mobilization by race are differences in activation of proximal signaling components of the PAR4 pathway including the PAR4 receptor, G_q turnover, and PLCβ activation, each of which is currently under investigation in the lab. Finally, novel signaling components are being investigated which may play an important role in racial differences of platelet reactivity. One such novel protein which was recently identified to be differentially expressed by race in human platelets, phosphatidylcholine transfer protein (PCTP),⁷ is actively being investigated to determine if differential expression of PCTP regulates PAR4-mediated platelet activation upstream of Ca²⁺ mobilization. Future investigation of a potential racial difference in relative risk for thrombosis in cardiovascular patients treated with dual antiplatelet therapy (aspirin and Plavix) will support the use of personalized treatment (based on genetic factors preserved in different racial groups) for the risk of a thrombotic events. The development of unique therapeutic approaches for prevention of thrombosis in each of these genetically unique populations may significantly decrease the observed morbidity and mortality due to occlusive thrombus formation and stroke following platelet activation.

This is the first in-depth investigation identifying the racial differences inherent in PAR4-mediated platelet activation. Platelet reactivity in blacks is significantly elevated compared to that observed in whites even in the presence of dual platelet inhibition (P2Y₁₂ antagonist and aspirin). While the current study suggests that white patients on dual anti-platelet therapy may be better protected from thrombotic events compared to their black counterparts, this hypothesis will need to be confirmed in patients taking dual anti-platelet therapy (COX-1 and P2Y₁₂ inhibitors). This study is timely as the PAR1 antagonist vorapaxar has recently been approved for the prevention of MI. In the presence of a PAR1 antagonist, thrombin is only able to activate platelets via PAR4. As more data on the PAR1 antagonist become available, it will be important to determine if black patients taking PAR1 antagonists, in addition to COX and P2Y₁₂ inhibitors, are equally protected from thrombotic events as white patients. Recent work including the current study suggests that platelets from black individuals are hyper-responsive to PAR4 stimulation. Thus, the development of a PAR4 antagonist may represent a targeted protection from thrombosis and MI in particular segments of the population at risk for cardiovascular diseases.

Significance.

Black individuals are at an increased risk of mortality from thrombotic diseases relative to whites. This study demonstrates that platelets from black donors have a lower threshold of activation to PAR4 stimulation relative to platelets from white donors due to enhanced activation of the biochemical components of G_q pathway including Ca²⁺ mobilization, Rap1 activation, and αIIbβ3 activation. This work suggests that current anti-platelet therapies may provide decreased protection against thrombotic diseases in black individuals compared to whites in response to PAR4 stimulation by thrombin.

Abbreviations

AA

arachidonic acid

ADP

adenosine diphosphate

AP

activating peptide

Ca²⁺

calcium

COX

cyclooxygenase

MFI

mean fluorescence intensity

MI

myocardial infarction

PAGE

polyacrylamide gel electrophoresis

PAR

protease-activated receptor

PCTP

phosphatidylcholine transfer protein

PKC

protein kinase C

PPP

platelet poor plasma

PRP

platelet rich plasma

TxA₂

thromboxane

SDS

sodium dodecyle sulfate