Discrete Role for Cytosolic Phospholipase A₂α in Platelets

Abstract

Among several different types of phospholipase A₂ (PLA₂), cytosolic PLA₂ (cPLA₂)α and group IIA (IIA) secretory PLA₂ (sPLA₂) have been studied intensively. To determine the discrete roles of cPLA₂α in platelets, we generated two sets of genetically engineered mice (cPLA₂α^−/−/sPLA₂-IIA^−/− and cPLA₂α^−/−/sPLA₂-IIA^+/+) and compared their platelet function with their respective wild-type C57BL/6J mice (cPLA₂α^+/+/sPLA₂-IIA^−/−) and C3H/HeN (cPLA₂α^+/+/sPLA₂-IIA^+/+). We found that cPLA₂α is needed for the production of the vast majority of thromboxane (TX)A₂ with collagen stimulation of platelets. In cPLA₂α-deficient mice, however, platelet aggregation in vitro is only fractionally decreased because small amounts of TX produced by redundant phospholipase enzymes sufficiently preserve aggregation. In comparison, adenosine triphosphate activation of platelets appears wholly independent of cPLA₂α and sPLA₂-IIA for aggregation or the production of TX, indicating that these phospholipases are specifically linked to collagen receptors. However, the lack of high levels of TX limiting vasoconstriction explains the in vivo effects seen: increased bleeding times and protection from thromboembolism. Thus, cPLA₂α plays a discrete role in the collagen-stimulated production of TX and its inhibition has a therapeutic potential against thromboembolism, with potentially limited bleeding expected.

Introduction

Platelets are required for hemostasis but also contribute in the pathogenesis of thrombotic and inflammatory diseases. A discrete therapeutic change in their properties is needed to maintain safety. Lipid mediators generated from phospholipid membranes, platelet-activating factor, or eicosanoids derived from released arachidonic acid (AA)*are thought to play an important role in their activation (1–4). The inhibition of an AA modifying enzyme, cyclooxygenase, by aspirin continues to be the single most successful therapy at preventing thrombotic disease (5). Thromboxane (TX)A₂ has been most clearly linked to function with the reports of genetic defects causing bleeding tendencies (6–8). There are potentially multiple classes of phospholipase A₂ (PLA₂) that may be involved in AA release: group IIA (IIA) secretory PLA₂ (sPLA₂), cytosolic PLA₂s (cPLA₂s), group V sPLA₂, calcium-independent PLA₂s, and group X sPLA₂ (9–13). As well as through the sequential action of phospholipase C (PLC), diglyceride lipase, and monoglyceride lipase, AA can be generated from phosphatidylinositol. However, cPLA₂α, acting primarily on phosphatidylcholine, is thought to be the main enzyme involved in AA release on the activation of platelets. The discovery of paralogues, β and γ forms of this enzyme, in addition to the previously cloned cPLA₂α form, has added further complexity (14–16).

Platelet exposure to matrix components such as collagen necessitates a rapid response. In a complex multistage process platelets change shape, degranulate, and then aggregate (17). The binding of platelets to collagen has been reported to involve at least two receptors. Initial binding is to an integrin, α₂β₁, which can bind at high sheer rates and is reported to activate AA release by PLA₂ enzymes. In vivo, this binding is likely followed by the subsequent binding of collagen to glycoprotein VI (18). Other platelet activators, thrombin, TXA₂, ADP, and platelet-activating factor, act through G protein–coupled receptors on the surface of platelets. The blocking of G protein signaling may effectively inhibit platelet aggregation induced by collagen interfering with the accelerating effects of TXA₂ and ADP released after collagen binding and signaling.

The study of the enzymes involved in AA release is restricted by the unavailability of highly specific inhibitors to the various PLA₂s. Mice lacking both cPLA₂α (engineered) and sPLA₂-IIA (spontaneous) were previously generated to study the role of cPLA₂α in vivo (19, 20). Backcrossing these mice into the C3H/HeN mice with a working sPLA₂-IIA gene creates a strain with only the cPLA₂α gene inactivated. Both strains of mice were generated in our laboratory and studied to question the specific role of cPLA₂α in platelet activation. In vitro studies were conducted with the simultaneous measurement of degranulation by ATP release and platelet aggregation through impedance allowing the evaluation of the continuous effects of the loss of these enzymes (21, 22). Bleeding times and a thromboembolism model were used to study the effects in vivo.

Our results support a key role for cPLA₂α in the production of TXA₂ in platelets but suggest a basic redundancy in its production from other mechanisms. We also found that ADP or TXA₂ activation of platelets appears independent of cPLA₂α and likely leads to the involvement of other PLA₂s. In vivo studies suggest that a specific cPLA₂α inhibitor, in addition to an antiallergic/antiinflammatory effect and a growth suppression effect on intestinal polyp (19, 20, 23–31), will therefore have an antivasoconstrictive effect by dramatically reducing the amount of TXA₂ produced.

Materials and Methods

Reagents.

1 mg/ml equine type I collagen, 1 mM ADP, ATP standard, and Chrono-lume reagent, a bioluminescent luciferase mixture, were purchased from Chrono-log Co. Before use, U46619, a TX receptor agonist in methyl acetate, was evaporated and diluted in PBS (Cayman Chemical). SQ29548, a highly specific TXA₂ receptor antagonist, was dissolved in ethanol for storage and then evaporated and diluted in PBS just before experiments were performed (Cayman Chemical). Indomethacin (Sigma-Aldrich) was dissolved in 0.1 M sodium bicarbonate before use. 12(S)-hydroxyeicosatetraenoic acid (HETE) was purchased from Cayman Chemical.

Mice.

Inbred mice were purchased from Central Laboratories for Experimental Animals Japan. As previously described (19), the cPLA₂α gene was disrupted by replacing part of an internal exon with a PGK-neo cassette. The mice generated (F2 of C57BL/6J and 129/Ola) were cPLA₂α^−/− and were also discovered to be congenitally defective in sPLA₂-IIA due to a frameshift mutation in the sPLA₂-IIA gene (32, 33). These mice were backcrossed to the C3H/HeN strain, which were gene sequenced to ensure the presence of a functional sPLA₂-IIA gene. Eighth and ninth generation backcrossed C3H/HeN mice were used. Twelfth generation backcrossed C57BL/6J mice were also used in the experiments. All mice were genotyped by a PCR method as previously described (19).

In Vitro Platelet Responses.

20–48-wk-old littermate backcrossed mice matched for sex were studied in pairs (cPLA₂α^+/+, cPLA₂α^−/−) to exclude any potential bias. Mice were anesthetized with 25 mg/kg pentobarbital and 25 mg/kg ketamine, and ∼1 ml of whole blood was immediately collected in 0.35% (wt/vol) sodium citrate after cardiac puncture. After 30 min, 100 μl whole blood was diluted with 800 μl normal saline and 100 μl Chromo-lume reagent. Aggregation as measured by change in impedance and ATP release from dense granules were monitored continuously by luminometry (Chrono-Log Co.). To allow for comparison between the experiments, aggregation/impedance was adjusted to measure a set change in resistance by changing the baseline and height of a standard impulse. A 2-nM dose of ATP was added at the end of each measurement to standardize ATP. If the baseline measurement of ATP indicated the presence of marked platelet degranulation during cardiac puncture, the experiment was terminated. Continuous measurements were made electronically at every 25 ms, but summarized to the second for analysis. Reagents were added only after 2 min of a flat baseline in both measurements and the ATP level was <0.1 nM.

TXA₂ Assay.

After 9 min of reaction, 100 μl of the aliquot was placed on ice. Samples were then spun at 1,000 g for 5 min and the supernatant was frozen at −70°C until analysis. For each mouse, 50 μl of the sample was saved to measure the baseline levels before stimulus. TXB₂, a nonenzymic hydrolyzed product of TXA₂, was measured by an enzyme immunoassay kit (Cayman Chemical).

12-HETE Assay.

Quantification of 12-HETE was performed by reversed phase HPLC (system GOLD; Beckman Coulter) using a solvent of acetonitrile/methanol/water/acetic acid, 350:150:250:1 (2, 34). Flow rate was 1 ml/min and eluent was monitored at 235 nm. Blood was collected from incisions on femoral vessels of anesthetized mice (25 mg/kg pentobarbital and 25 mg/kg ketamine). After 30 min of incubation at room temperature to allow clotting, serum samples were collected by centrifugation at 500 g for 5 min. Samples were extracted with ethylacetate after acidification to a pH of ∼5.0 with 0.5 vol of 0.1% formic acid, dried on a centrifuge concentrator, dissolved in the HPLC solvent, and analyzed. To normalize the extraction rate, 0.5 nmol 12(S)-HETE was added to selected samples before extraction and the difference of the area was calculated.

Bleeding Time.

Adult mice were restrained in the upright position and the tail was cut 2–3 mm from the tip. The tail was then immersed in saline at 37°C and the bleeding time was defined as the time point at which all visible signs of bleeding from the incision had stopped, or at 10 min (35). Because of a significant difference between males and females in knockout C3H/HeN, only females were used in later studies. 10 mg/kg indomethacin was injected intraperitoneally 1 h before the bleeding times were tested in age-matched female mice.

Thromboembolism Test.

In mice anesthetized with 80 mg/kg sodium pentobarbital, a collagen–ADP mixture (4 ml/kg of a saline-based solution containing 250 mg/ml collagen and 200 μM ADP) was injected into the jugular vein. Survival was evaluated 1 h after injection (36, 37). The dose was pretested to ensure 100% killing of wild-type mice (38). One pair of mice were killed and intubated 2 min after injection. 4% formalin in PBS was injected into the lung, and then the heart and lung were dissected together and placed in 10% formalin in PBS. Serial sections were cut and stained with hematoxylin and eosin for examination. A blindfolded investigator counted 20 areas at 400× for each mouse to determine the percentage of clotted vessels.

Statistics.

All values are expressed as mean ± SD unless stated otherwise. Two-tailed t tests were used to test the significance on continuous data and multiple regression was used to calculate the P-values on nominal data and in multivariable analysis (Quattro Pro).

Results

Degranulation and Aggregation to Collagen.

C3H/HeN cPLA₂α^−/− sPLA₂-IIA^+/+ mice show a delay in degranulation as measured by ATP release in response to collagen (Fig. 1 a). This accompanies a more concave impedance curve, indicating a slowing in the acceleration phase of platelet aggregation. Aggregation achieved at 9 min was preserved in cPLA₂α^−/− platelets. The time to achieve peak ATP release (Fig. 2 , left, P = 0.013) and the time to reach 50% of aggregation achieved within 9 min (Fig. 1 a; cPLA₂α^+/+, 227 ± 43.7 s vs. cPLA₂α^−/−, 268 ± 42.8 s; P = 0.042) is significantly delayed. Focusing on the first 20 s of the reaction, it appears that the loss of cPLA₂α starts to have an effect on the rate of degranulation at about the 20-s mark (Fig. 1 a). Compared to the changes in response to collagen, the reaction to U46619, a TX agonist, and ADP, both of which work through G protein–mediated receptors, appears to be unchanged (Fig. 1, b and c). C57BL/6J cPLA₂α^+/+ sPLA₂-IIA^−/− mice appear to have the same shaped curve as C3H/HeN cPLA₂α^+/+ sPLA₂-IIA^+/+ mice (Fig. 1 d). C57BL/6J cPLA₂α^−/− sPLA₂-IIA^−/− mice also show a significant delay in the degranulation peak (Fig. 2), and a more concave aggregation curve. The times to reach 50% of aggregation were as follows: cPLA₂α^+/+ sPLA₂-IIA^−/−, 178 ± 14.6 s versus cPLA₂α^−/− sPLA₂-IIA^−/−, 255 ± 67.0 s; P = 0.021. An indirect comparison of the changes between C3H/HeN and C57BL/6J mice using time to peak ATP can be attempted. There is an indication of an additional step delay due to the additional loss of sPLA₂ when compared with cPLA₂α loss alone (Fig. 1, a and d, and Fig. 2). The effect of other factors cannot fully be excluded due to the differences in the mouse strains used.

Figure 1.

Effect of cPLA₂α on platelet activation. ATP release (left axis) as monitored by luciferase activity is seen on the top of each graph. The first 20 s of the reaction is magnified above. Platelet aggregation (right axis) as measured by impedance is at the bottom. In each case, averaged data from wild-type littermates (^+/+) are paired with sex-matched cPLA₂α-deficient mice (^−/−). (a) C3H/HeN mice platelet reaction to collagen (n = 9). (b) C3H/HeN reaction to U46619 (n = 7). (c) C3H/HeN reaction to ADP (n = 6). (d) C57BL/6J reaction to collagen (n = 4).

Figure 2.

Time to peak ATP after collagen. The mean of individual times to maximum ATP level of each mouse is used in Figs. 1 and 4 plotted by strain, cPLA₂α status, and TXA₂ antagonist, SQ29548. Error bars are SD above the mean and P-values stand for significant differences.

TX Levels.

Compared to the small difference in degranulation and aggregation in vitro (Figs. 1 and 2), there is a marked, significant decrease in the amount of TXB₂ production as detected after collagen activation between cPLA₂α^+/+ and cPLA₂α^−/− mice in both C3H/HeN and C57BL/6J strains (Fig. 3) . The absolute difference in the C57BL/6J mice is larger (3.5 vs. 1.4 ng/ml), which is consistent with the ATP and aggregation curves. With the increase in TXA₂ upon stimulation, it is also clear that a limited amount of AA is being released in both strains of cPLA₂α^−/− mice, indicating the presence of another PLA₂ or an alternative AA-generating pathway. In accord with the aggregation and degranulation data (Fig. 1), there is little difference in TXB₂ production in response to ADP between C3H/HeN wild-type and cPLA₂α-deficient mice, suggesting a cPLA₂α-independent mechanism for TXA₂ production with ADP as a stimulus. Similarly in C57BL/6J platelets stimulated with ADP, there was little difference in ATP release and aggregation (unpublished data), and TXB₂. Therefore, an AA-releasing mechanism, possibly another PLA₂(s) or PLC–diglyceride lipase system independent of both cPLA₂α and sPLA₂-IIA, is present in platelets upon the stimulation of G protein–coupled receptor ligands.

Figure 3.

TXB₂ produced after platelet stimulation. Mean change in TXB₂ levels of mice pairs in Fig. 1. Open columns, cPLA₂α^+/+ mice; shaded columns, cPLA₂α^−/− mice. Because of the log scale, actual mean is given above the columns, error bars are SD above the mean, and P-values stand for significant differences.

Effect of a Specific TX Antagonist.

To confirm the presence of additional specific TXA₂ activity generated by cPLA₂α/sPLA₂-IIA, a specific TXA₂ receptor antagonist, SQ29548, was used (39). Platelet activation appears both TXA₂-dependent and -independent, as SQ29548 was unable to completely block activation in all cases (Fig. 4) . Upon examining the TXA₂-dependent component, the results on C3H/HeN cPLA₂ ^−/− sPLA₂-IIA^+/+ mice and C57BL/6J cPLA₂ ^−/− sPLA₂-IIA^−/− in Fig. 4, c and d, confirm the presence of an alternative redundant enzyme(s) because a persistent inhibiting effect is seen in both mouse strains. In addition, SQ29548 inhibited degranulation and aggregation slightly more in the C57BL/6J mice, where the sPLA₂-IIA enzyme activity is missing, than in the C3H/HeN mice (Fig. 4, compare a and c, and b and d). A hint of the additional effect of sPLA₂-IIA is also seen when all the times to peak ATP results are graphed (Fig. 2). There appears to be a stepwise delay as successive enzymes are lost or TXA₂ activity is blocked. Statistically, however, multiple regression of the factors that influence time to peak ATP demonstrates an independent significant effect of the loss of cPLA₂α and the inhibitor SQ29548. The effect of the mice strain (the loss of sPLA₂-IIA) did not reach statistical significance. Therefore, in these studies we can only conclude that cPLA₂α^−/− platelets have an alternative AA-generating mechanism to cPLA₂α and sPLA₂-IIA. However, it is interesting to see that as expected, the effect of the inhibitor is smaller in the C57BL/6J strain with only a possible delay in the initiation of the reaction seen (Fig. 4, c and d). There is also a suggestion that the inhibitor effect of SQ29548 occurs earlier than at 20 s when compared with the effect of the loss of cPLA₂α as seen in Fig. 1.

Figure 4.

Effect of a TXA₂ receptor antagonist, SQ29548, on platelet activation. ATP release (left axis) as monitored by luciferase activity is seen on the top of each graph. The first 20 s of the reaction is magnified above. Platelet aggregation (right axis) as measured by impedance is at the bottom. In each case, responses to collagen are compared with and without SQ29548 from pooled data from wild-type (^+/+) or cPLA₂α-deficient mice (^−/−). (a) C3H/HeN cPLA₂α^+/+ mice (n = 3), (b) C57BL/6J cPLA₂α^+/+ mice (n = 3), (c) C3H/HeN cPLA₂α^−/− mice (n = 2), and (d) C57BL/6J cPLA₂α^−/− mice (n = 2).

Serum 12-HETE Levels.

Next, we determined the contribution of cPLA₂α on the production of 12-HETE, another AA metabolite. As shown in Fig. 5 a, elution of 12-HETE was observed at 11.9 min on HPLC analysis. The absorbance spectrum was indistinguishable from authentic 12(S)-HETE, and coelution was also observed when they were mixed (not depicted). As shown in Fig. 5 b, the calculated concentration of 12-HETE in serum from C3H/HeN cPLA₂α^−/− adult female mice was 0.72 ± 0.28 μM (n = 14), significantly lower than cPLA₂α^+/+ (2.4 ± 1.5 μM; n = 13; P < 0.001) and cPLA₂α^+/− (1.8 ± 0.83 μM; n = 15; P < 0.05). A similar significant difference was observed for C57BL/6J adult male mice: cPLA₂α^+/+ (3.2 ± 1.6 μM; n = 6) versus cPLA₂α^−/− (0.44 ± 0.25 μM; n = 6; P = 0.01; Fig. 5 b).

Figure 5.

Serum 12-HETE content. (a) Representative HPLC chart. ^+/+, a cPLA₂α^+/+ mouse (3.6 μM); ^−/−; a cPLA₂α^−/− mouse (1.0 μM). (b) Mean levels of 12-HETE in serum. Open columns, cPLA₂α^+/+ mice; hatched column, cPLA₂α^+/− mice; shaded columns, cPLA₂α^−/− mice. Error bars are SD above the mean and P-values stand for significant differences.

Marked Changes in Bleeding Time.

Compared to the in vitro aggregation results, the differences in bleeding time correlate with TXB₂ levels. Bleeding times in both C3H/HeN and C57BL/6J mice were significantly increased (Fig. 6 a). However, because of a marked difference between male and female mice (unpublished data), only female mice were used for comparison between strains and interventions. Bleeding times, like the in vitro aggregation results, show that the cPLA₂α^−/− C57BL/6J mice have longer bleeding times than C3H/HeN females and cyclooxygenase inhibition with indomethacin leads to a similar increase in bleeding time.

Figure 6.

In vivo effects of cPLA₂α. (a) Bleeding time. Open columns, cPLA₂α^+/+ female mice; shaded columns, cPLA₂α^−/− female mice. C3H/HeN mice (cPLA₂α^+/+, n = 15; cPLA₂α^−/−, n = 10), C57BL/6J (cPLA₂α^+/+, n = 7, wild-type nonlittermates; cPLA₂α^−/−, n = 7), and C3H/HeN mice treated with indomethacin (six mice in each group stratified by bleeding time). Error bars are SD above the mean and P-values for significant differences between cPLA₂α^+/+ and cPLA₂α^−/− are shown in each case. (b) Death in thromboembolism test of C3H/HeN littermate mice matched for sex and eight or ninth generation backcrossed. (c) Lung histology of cPLA₂α^−/− and cPLA₂α^+/+ hematoxylin and eosin stain. In cPLA₂α^−/− mice at a low magnification (top) there are multiple platelet clots seen, causing red blood cell congestion in small and medium vessels. At a higher magnification (bottom), draining veins can be seen with platelet clots. In cPLA₂α^+/+ mice at a low magnification (top), smaller vessels appear to be clotted. At a higher magnification a muscular artery is seen with platelet clots. B, bronchi; LN, lymph node. Bars, 100 μM.

Protective Effect of cPLA₂α Loss on Thromboembolism.

In the thromboembolism test there was an increase in survival that is significant in cPLA₂α^−/− mice (Fig. 6 b). After the dissection of the heart and lungs together, the cPLA₂α^−/− lungs were of a much darker red than the cPLA₂α^+/+, suggesting increased vasoconstriction in the presence of cPLA₂α. However, both were engorged with blood compared with normal lungs due to clots. Blinded counts show an equal percent of clotted vessels in the wild type and knockout (cPLA₂α^+/+, 76% vs. cPLA₂α^−/−, 82%). The percentage is similar to previous studies (37). In cPLA₂α^+/+ mice, small vessels were mostly clotted with evidence of arterial constriction and platelet clots in large arteries (Fig. 6 c, top and bottom right). In cPLA₂α^−/− mice, perhaps because of the lack of TXA₂-induced arterial constriction, platelet clots were allowed to reach larger collecting veins (Fig. 6 c, top and bottom left). The large number of platelet clots seen in knockout mice indicates that platelets are able to aggregate well, as suggested by the in vitro studies. The collection of clots in the venous side of the pulmonary system in mice lacking cPLA₂α appears to be more forgiving than clotting of the arterial side of the pulmonary artery system, which explains the reduction in mortality.

Discussion

By using platelets of two sets of genetically engineered mice, we found that cPLA₂α plays a critical role in TXA₂ production but a relatively redundant autocrine role in platelet aggregation due to an alternative AA-releasing mechanism. The lack of cPLA₂α leads to a dramatic loss of TXA₂ production from collagen-activated mouse platelets in vitro. This loss contrasts to a small but significant delay in ATP release and a small but significant loss in the acceleration of platelet aggregation. The TXA₂ results, however, correlate well with a marked increase in bleeding time in mice lacking cPLA₂α. This discrepancy is best explained if the vasoconstrictive effect of TXA₂ is considered. The test of bleeding time transects large veins and arteries in the mice tail. The veins, in particular, could be seen bleeding profusely in some cPLA₂α^−/− males where the diameter of the tail is considerably larger than that of the female, whereas in cPLA₂α^−/− mice bleeding could stop abruptly at 60–90 s due to vasoconstriction. The need for more vasoconstriction normally induced by TXA₂ would help explain the difference in bleeding time between the cPLA₂α^−/− C3H/HeN males (large diameter vessels) and females (smaller diameter vessels; unpublished data). This explanation fits well with the biological effects of TXA₂ inhibition with aspirin. Acute vascular syndromes are being prevented but with well-preserved aggregation at low intermittent dosing, i.e., inhibiting much, but not all, of the TXA₂. Small amounts of TXA₂ might be required early by platelets to activate themselves effectively, whereas the larger amounts produced mostly by cPLA₂α are intended for vasoconstriction. All of the current results support a dual function for TXA₂ and a discrete role for cPLA₂α in mouse platelets. (a) There are low levels of TXA₂ in both strains of knockout mice and therefore relatively well-preserved aggregation. (b) ADP, TXA₂ (Fig. 1, b and c, and Fig. 3), and probably other G protein–coupled receptor ligands, independent of cPLA₂α, stimulate TXA₂ production, suggesting a redundant source of low level AA release. (c) In both strains of mice, a specific TXA₂ antagonist appeared to extend the delay in aggregation in proportion to the level of TXA₂ expected to be generated and also appeared to have an earlier effect. (d) There is a stepwise increase in bleeding times due to the blocking of successive enzyme loss between cPLA₂α^−/− and cPLA₂α^−/−/sPLA₂-IIA^−/− mice, similar to the in vitro aggregation pattern. (e) During thromboembolism, the preservation of the ability to form platelet clots in cPLA₂α^−/− mice with decreased lethality is probably due to the modification of vascular effects.

These results suggest that although cPLA₂α is responsible for the vast majority of TXA₂ production in activated platelets after collagen stimulation, there are redundant enzymes in platelets that can maintain basic platelet aggregation with relatively small amounts of TXA₂. The loss of this baseline autocrine TXA₂ effect leads to a more profound loss of platelet function. At least two independent mechanisms of TXA₂ production are apparent in mouse platelets with different PLA₂ enzymes, production levels and physiological effects. Additional support for this is the log fold difference in the dose of TXA₂ agonist required for smooth muscle contraction and platelet activation, even though the TXA₂ receptors appear to be the same (40, 41). There is mounting evidence for the dual role, an intracellular role and an intercellular/extracellular role, for any given lipid mediator (42). The cPLA₂α enzyme appears to play a critical part in generating lipid mediators for extracellular roles in platelets. It is also possible that platelets can utilize AA provided by serum lipids and neighboring cells.

The loss of cPLA₂α also appears to be important in the release of AA needed to produce other lipid mediators that may augment platelet aggregation/TXA₂ production (2). The loss of 12-HETE production evident in mice without cPLA₂α provides another mechanism by which the absence of cPLA₂α could act. The amount of 12-HETE dependence on cPLA₂α during aggregation may also apply to 12-lipoxygenase activity in other cell types and could contribute to the antiatherosclerosis/antiangiogenesis effect of cPLA₂α inhibition (43–46).

The delay in α granule release, as reflected in ATP, associates with and precedes the delay in aggregation and therefore may play a mechanistic role. There appears to be a more generalized delay in degranulation in cells missing cPLA₂α (25, 26). The delay in degranulation is not present with a TX agonist or ADP and is reproduced with a specific antagonist, which suggests that a reduction in the pace of the reaction is the only explanation needed for the delay in ATP release in these studies.

Rather than representative data, electronic summarized graphs are chosen in our experiments to block out the multiple factors that can influence platelet studies in mice. Variations in activation and platelet numbers during cardiac puncture cannot be easily standardized. As an alternative to the current approach, dilution of whole blood to a standard platelet count would add the variation caused by the dilution of plasma factors. During preliminary studies on third generation mice, small differences in platelet response were seen. Therefore, steps such as blocking for age and sex were used to decrease all other possible sources of variation. The presence of similar results in both strains of mice and the complimentary findings with the use of a specific antagonist to TXA₂ validates the approach. In vitro platelet data must, however, be viewed with some skepticism (47). In mice where careful controlled collection of blood is difficult, special care to correlate in vitro and in vivo experiments is needed. Although the use of different strains of mice help strengthen the generalization of the results, the comparisons between the two strains have to be carefully interpreted. The base levels of TXA₂ and ATP levels between C3H/HeN and C57BL/6J in response to collagen are different. Without a C57BL/6J mouse with a functional sPLA₂-IIA as a control, conclusions on the role of this enzyme in platelets cannot be certain. The addition, however, of studies using the C57BL/6J does show that sPLA₂-IIA is not the only alternative AA-generating enzyme to cPLA₂α in platelets.

Previous platelet studies with engineered mice put these new findings in context. Mice with Gαq deficiency have severe loss of platelet functions with little evidence for redundant processes for this key protein (48). In knocking out P2Y1, an ADP receptor, evidence was found for a second, partially redundant receptor that was recently cloned (36, 49, 50). The surprise finding, the disruption of CD39, which is an ATP diphosphohydrolase, was an inhibition of platelet aggregation (51). AA released by PLA₂s may also be acted upon by 12-lipoxygenase to form 12-HETE. Knockout studies suggest that this might be a factor that plays a unique role in ADP-stimulated platelets (2). In TXA₂ receptor knockout mice, findings very consistent with the current results were found (1). The bleeding times were also prolonged with the loss of TXA₂ receptor, which contrasted with human receptor deficiency. However, the test for bleeding time in humans and that used in mice is very different. The vasoconstriction caused by TXA₂ may have a significant effect on the bleeding times measured on mice tails compared to the human tests where aggregation in small skin vessels is tested. A delay in collagen activation compared to ADP activation was noted similarly to our paper. The current, more sensitive studies confirm a delay in the initiation of aggregation but also suggest that a stepwise loss of TXA₂ has effects on aggregation, with the complete loss of all TXA₂ having a more pronounced effect on aggregation. The current results cannot be directly compared with those of the TXA₂ receptor knockout because of different methodologies. It cannot be excluded, however, that an additional mechanism, which is TXA₂ receptor independent, is present. Studies involving multiple knockouts and specific inhibitors are needed to take the entire process apart.

The use of specific inhibitors to intracellular signaling proteins and knockout studies has provided some insights into how cPLA₂α might be regulated in platelets. First, G protein–coupled receptors to ADP, thrombin and TXA₂, appear to work through Gq proteins and activate PLC βs (48, 52, 53). Second, the recently cloned glycoprotein VI appears to require coactivation of the integrin α₂β₁ to initiate a reaction. That reaction involves lyn and fyn Src family kinases that affect p72 Syk, leading to a pathway through SLP-76 to PLCγ2 (17, 54–57). Third, the signaling though α₂β₁ involves actin polymerization and probably involves the activation of an Src kinase leading to a mitogen-activated protein kinase–dependent cPLA₂ activation (58–61). Therefore, platelet activation appears to involve at least three different pathways that interact with one another. cPLA₂α activation would appear to be related to the pathway through integrin α₂β₁ and an alternative mechanism, most likely another PLA₂(s), through G protein–coupled receptors (ADP or TXA₂, etc.). The picture that emerges is that platelet activation is a complex multichanneled process involving multiple surface receptors and intracellular signaling pathways that mostly behave in vivo with increasing intensity. The loss of any particular protein may thus have a broad or distinct effect depending on its unique placement in a complex scheme.

Although these results in mouse platelets provide insights into human platelet function, in vivo use of specific inhibitors of cPLA₂α (62) and sPLA₂-IIA are needed to confirm these results in humans.