Coronary artery disease is accelerated in chronic spinal cord injury (SCI). Because prostacyclin (PGI2) may retard atherogenesis through its inhibitory effects on platelet function, the role of PGI2 on SCI platelets was determined. The SCI platelets were neither hypersensitive to aggregating agonists nor resistant to the inhibitory effect of PGI2, but PGI2 failed to inhibit platelet-stimulated thrombin generation and the release of platelet-derived growth factor (PDGF) in SCI. Because thrombin and PDGF are atherogenic mitogens, the generation of these mitogens was investigated. Both the release of PDGF and thrombin generation in SCI platelets were higher when compared with control (n = 12). Treatment of non-SCI platelets with 100 nM PGE1 (a stable probe of PGI2) inhibited the release of the mitogens by 90% (P< 0.001), with no effect on SCI platelets. Scatchard analysis of prostaglandin E1(PGE1) binding showed a 70% decrease of PGI2 receptors on the SCI platelet surface. Treatment of SCI platelets with insulin or Ca2+ channel blockers restored the PGI2-receptor number and “normalized” the inhibition of PDGF release and thrombin generation by PGI2.
- prostaglandin E1
- coronary artery disease
- platelet-derived growth factor adenosine 3′,5′,-cyclic monophosphate
coronary artery disease (CAD) is accelerated in individuals with chronic spinal cord injury (SCI), and the underlying mechanism is poorly understood (2, 18). Aggregation of platelets induced by agonists, such as ADP, thrombin, or epinephrine, is critical in normal blood coagulation (28, 32). Aggregation also results in the release of a platelet-derived growth factor (PDGF), which is a well-recognized potent mitogen for cells of mesenchymal origin. PDGF has been associated with the induction of apoptotic cell death, as well as a number of human diseases, including atherosclerosis, glomerulonephritis, and cancer (4, 33). Chronic SCI subjects may have an increased incidence of silent ischemia and thus undiagnosed CAD (2). Recent studies have demonstrated that the conversion from subclinical to overt CAD is due to endothelial injury at the site of stenosis by rupture or fissure of the atherosclerotic plaque (1). At the sites of endothelial injury, there is accumulation of several mediators that promote platelet aggregation and vasoconstriction, including thromboxane A2(TXA2), ADP, serotonin, activated thrombin, platelet-activating factor, and tissue factor (1,41).
Thrombin regulates hemostasis and thrombosis, is the most potent stimulator of platelet aggregation, and has an essential role in blood coagulation. Thrombin is also a potent mitogenic agent for arterial smooth muscle cells, inducing the proliferation of human mesengial cells that induce the mRNA synthesis encoding PDGF, and it has been implicated in atherosclerotic plaque formation (6, 37). The effects of thrombin and PDGF that are mediated through the interaction with platelets are inhibited by prostacyclin (PGI2) binding to specific receptors on the platelet surface, which activates membrane-bound adenylate cyclase and increases cAMP (3, 8, 12, 14, 21, 29, 36, 42). The increase of intraplatelet cAMP inhibits platelet aggregation, platelet-stimulated thrombin generation, and the release reaction (12,20, 42), which includes the release of PDGF. Furthermore, PGI2 almost completely prevents agonist-induced Ca2+ mobilization from intracellular stores and inhibits store-dependent Ca2+ influx; these effects are mediated by cAMP protein kinase (9). PGI2, through its aforementioned inhibitory properties, is believed to exert a significant beneficial effect on the prevention of atherosclerosis (17, 22).
Although the occurrence of increased incidence of premature cardiovascular disease among persons with chronic SCI is well documented (2, 18), the underlying pathophysiological molecular events are unknown. Recently, we have reported for the first time the appearance of a novel circulating antibody in chronic SCI individuals, which specifically blocks the high-affinity PGI2 receptors on the platelet surface without affecting either the low-affinity PGI2 receptors on the same platelet or the stimulation of adenylate cyclase through the binding of the agonist to its low-affinity receptors on platelet membranes (19).
This report demonstrates that the impaired inhibitory effect of the prostanoid in thrombin-induced PDGF release in platelets and platelet-mediated stimulation of thrombin generation in chronic SCI were due to the impaired binding of PGI2 to its high affinity. In these platelets, the impaired inhibitory effect of PGI2 was restored by the upregulation of PGI2 high-affinity receptor number with physiological concentrations of insulin or calcium channel blockers, in vitro or in vivo.
MATERIALS AND METHODS
Patient selection. Patients with chronic SCI (n = 12) between the ages of 32 and 65 yr were age-matched with 12 able-bodied controls ranging in age from 28 to 58 yr.
To study the effect of calcium channel blockers on thrombin generation and PDGF release in vivo, six SCI subjects with a well-documented history of chronically elevated blood pressure (>145/95 mmHg) without any apparent underlying cause who were followed at the outpatient SCI clinic were recruited for the study. Mean age was 45.6 ± 8 yr. Each patient had been treated with one or more hypertensive agents. Medications included calcium channel blockers (verapamil HCl was obtained from Searle and administered at a dose of 40 mg 3 times/day, and nifedipine was obtained from Pfizer and given at a dose of 10 mg 3 times/day). The patients were asked to discontinue all hypertensive medications 2 wk before the study; during that period, they were closely monitored for any evidence of accelerated or malignant hypertension. Patients in whom the withdrawal of antihypertensive agents was considered hazardous were excluded from the study.
After the basal blood samples were collected, the patients were instructed to resume their hypertensive medications, and after 24 h, a second blood sample was collected. None of the volunteers had taken aspirin 2 wk before the study. All volunteers had no known history of CAD or diabetes mellitus. The protocol was approved by the Institutional Review Board for Clinical Research, Veterans Affairs Medical Center, Bronx, NY, before recruitment of the subjects.
Chemicals. Insulin (HumulinR) was purchased from Eli Lilly (Indianapolis, IN). Prostaglandin E1(PGE1), PGI2, cAMP, and all other chemicals were obtained commercially (Sigma, St. Louis, MO). All chemicals used were of analytical grade.
Preparation of platelet-rich plasma, platelet aggregation, and washed platelets. Blood samples (40–50 ml) were collected from the subjects with 19-gauge siliconized needles in plastic tubes containing sodium citrate (0.013 M final concentration). Platelet-rich plasma (PRP) was prepared by centrifuging the blood samples at 200g for 15 min at 23°C. Platelet counts were determined in a Coulter counter. Aggregation of platelets by agonists, such as ADP, thrombin, or collagen and the inhibition of aggregation by PGE1 were studied in a aggregometer (Chronolog, Broomall, PA) by the stirring of PRP at 1,200 rpm at 37°C (24).
Platelets were washed with Tyrode buffer (without Ca2+), pH 7.5, containing 1.0 mM EDTA as described (24). After this, the platelets were washed twice with 2 vol of Tyrode buffer and suspended in the same buffer, without EDTA, containing 5.0 mM MgCl2(∼7 × 108 cells/ml).
Platelet PGI2/PGE1-receptor assay. Because PGI2 and PGE1 bind to the same receptor on the platelet membrane (17, 20, 22), and radiolabeled PGI2 as free-acid form is not yet commercially available, [3H]PGE1([5,6-3H(N)]PGE1; specific activity, 55 Ci/mmol; New England Nuclear, Boston, MA) was used as a probe to assess PGI2-receptor activity.
Approximately 2 × 108platelets in Tyrode buffer, pH 7.5, containing 5 mM MgCl2 were incubated with 3 nM [3H]PGE1(30,000 counts/min) in a total volume of 200 μl at 23°C for 15 min to attain equilibrium. The platelet suspension was filter washed as described (16, 18, 21, 23, 24). The nonspecific binding was determined by adding excess (15 μM) unlabeled prostanoid to the assay mixture. The specific binding was calculated (23).
For the studies in which an effect of insulin was investigated, PRP was incubated at 23°C with insulin (200 μU/ml; HumulinR) for 2.5–3 h and used for PGI2/PGE1binding (23).
Scatchard analysis of [3H]PGE1binding to platelets. The dissociation constants (K d) and the capacities (n, receptor) of the PGI2/PGE1receptors were determined by Scatchard analysis (35). Platelets were incubated with 3 nM [3H]PGE1plus 0–3 μM unlabeled PGE1for 15 min at 23°C (16, 18, 21, 23, 24). The dissociation constants and capacities were obtained from nonlinear regression analysis of equilibrium binding by a noniterative, least-square algorithmic analysis with a microcomputer (Elsevier, BIOSOFT, Cambridge, UK).
cAMP assay. The basal level of cAMP and the increase of intracellular content by PGI2/PGE1in platelets were determined by the protein kinase-binding method (10,20). Washed platelets in Tyrode buffer, pH 7.5, containing 5.0 mM MgCl2 were incubated with 10 mM theophylline in a total volume of 200 μl for 2 min at 23°C. Ice-cold TCA (5%) was added to the mixture, and the formation of cAMP was determined (10, 23).
Thrombin generation. The rates of thrombin generation in PRP and platelet-poor plasma were measured by determining the recalcification time (13). Serially diluted plasma or PRP (0.2 ml) was incubated with 0.85% NaCl at 37°C. After 1 min, 0.1 ml of 0.025 M CaCl2 was added to the mixture at 37°C and the recalcification time was determined. To determine the effect of PGI2/PGE1(10 nM PGI2), insulin (200 μU/ml), or calcium channel blockers (10 μM verapamil or pimozide) on thrombin generation, PRP was incubated, at 23°C for 5 min, 2.5 h, or 30 min, respectively, and thrombin generation was determined (18).
Assay of plasma PDGF. PDGF is present in platelet granules and is released upon platelet activation. To measure levels of PDGF, platelet-poor plasma was collected for measurement. Typically, PRP aliquots were incubated with or without insulin (200 μU/ml) or calcium channel blockers (10 μM) or PGI2 (10 nM) for 2.5 h, 30 min, or 5 min, respectively. After incubation, the PRP aliquots were treated with optimal concentrations of thrombin and other agonists to induce 100% aggregation. Immediately after, the reaction was stopped by adding 5% TCA, the supernatant was collected by centrifugation at 8,000 g at 4°C, and the samples were stored at −20°C. The effect of insulin, calcium ion blockers, or PGI2/PGE1on the production of PDGF was determined by an ELISA system (BIOTRAK, Amersham).
Assay of TXA2 . TXA2 is unstable and readily converted to thromboxane B2. The production of TXA2 in platelets was determined by radioimmunoassay (kit from Amersham) of thromboxane B2.
Statistical analysis. Results are shown as means ± SD or means ± SE. The data were analyzed by paired or unpaired t-test, applied appropriately. Probability values of P< 0.01 were considered significant.
Effect of PGI2/PGE1on inhibition of platelet aggregation and cAMP formation in PRP. The agonist-induced aggregation of platelets from SCI subjects did not show any detectable hyperactivity when compared with normal controls. The minimum amount of ADP or thrombin needed for optimal aggregation of platelets from SCI subjects when compared with non-SCI controls was 4.1 ± 1.6 vs. 4.0 ± 1.2 μM and 0.2 ± 0.06 vs. 0.2 ± 0.04 U/ml, respectively. The inhibition of platelet aggregation by PGI2/PGE1was similar in the two groups. Furthermore, the increase of cAMP by PGI2/PGE1was not significantly different (Table1).
Effect of PGI2/PGE1on platelet-stimulated thrombin generation, secretion of PDGF and TXA2. Treatment of PRP from SCI subjects with 10 nM or even higher amounts (up to 100 nM) of PGI2/PGE1produced no inhibitory effect on thrombin generation time in contrast to that in control platelets. PDGF and TXA2 release was not affected in SCI platelets by treatment with PGI2/PGE1compared with normal non-SCI PRP (Table 2).
Effect of PGI2on thrombin-induced release of PDGF and TXA2 . Because the release of PDGF and TXA2 occurs in parallel with platelet aggregation, the release of PDGF and TXA2 by thrombin in the PRP of SCI subjects was compared with controls. PDGF levels were assayed by an ELISA system. The basal level of PDGF in SCI platelet-free plasma was threefold higher when compared with the normal level (6.41 ± 0.12 vs. 2.15 ± 0.12 pg/106 cells, Fig. 1). The basal TXA2 levels were 11 ± 1.38 and 8 ± 0.66 pg/0.1 ml. The thrombin-induced release of PDGF was also consistently higher in SCI when compared with non-SCI subjects (24.2 ± 1.2 vs. 15.11 ± 1.6 pg/106 cells); the release of TXA2 was also increased in SCI platelets (from 11 ± 1.38 to 46 ± 6 pg/0.1 ml). The high PDGF levels in SCI subjects suggested marked platelet activation. Pretreatment of platelets with 10–100 nM PGE1 (used as a stable probe of PGI2) inhibited the release of the mitogen (Fig. 1) and TXA2 (46 ± 6 vs. 5 ± 1 pg/0.1 ml) by 90% (P < 0.001) in control platelets. In contrast, similar treatment of platelets from SCI subjects had no such effect.
Effect of incubation of PRP with insulin from SCI and normal subjects on [3H]PGE1binding and on the inhibition of thrombin generation and PDGF and TXA2 release.Scatchard analysis of the binding of [3H]PGE2to SCI and control platelets showed a heterogeneous receptor population (i.e., one high-affinity, low-capacity PGI2-receptor population and one low-affinity, high-capacity PGI2-receptor population). In the SCI platelets, the number of high-affinity binding sites of the receptors was significantly decreased (48 ± 11 vs. 161 ± 40 sites/cell;P < 0.001) without a significant change in the affinity of the binding sites when compared with controls (Table 3). Because treatment of platelets with physiological levels of insulin has been shown to increase both high- and low-affinity PGI2/PGE1-receptor numbers without changing their affinities (20, 23) and PDGF-stimulated cells have been demonstrated to require insulin and insulin-like growth factors to grow (25), platelets from SCI subjects were incubated with insulin (200 μU/ml; Refs. 18, 23). After incubation, the binding of [3H]PGE1to the platelets was analyzed by Scatchard plot. In parallel experiments, the inhibitory effect of PGI2 on platelet-stimulated thrombin generation and TXA2 and PDGF release was determined (Table 3). Incubation of SCI platelets with insulin not only restored the high-affinity PGI2 binding sites (n1) to “normal ranges” (48 ± 11 to 142 ± 36 sites/cell) without any significant change in affinity but also resulted in the prostanoid-induced inhibition of thrombin generation and TXA2 and PDGF release. The PGI2/PGE1inhibition in these insulin-treated SCI platelets was similar to that of control platelets. Treatment of control non-SCI platelets with insulin resulted in an above baseline increase of both the high- and low-affinity PGI2-receptor numbers compared with the nontreated platelets (Table 3).
Effect of Ca2+ channel blockers on thrombin generation and TXA2 and PDGF release in SCI platelets. The PRP from SCI subjects was incubated with calcium channel blockers, verapamil, or pimozide (10 μM), at 37°C for 30 min, and the rate of thrombin generation and TXA2 and PDGF release was determined (Table 4). Incubation of SCI platelets with verapamil, like the effect of insulin, also restored the high-affinity PGI2 receptors to normal ranges (n1= 46 ± 20 vs. 157 ± 30 sites/cell; n2 = 1,332 ± 261 vs. 2,088 ± 315 sites/cell) without any significant change in the affinity (K d1 = 7.21 ± 0.14 vs. 5.2 ± 1.5 nM andK d2 = 1.5 ± 0.2 vs. 1.8 ± 0.5 μM). Treatment of PRP from SCI subjects with calcium channel blockers corrected the PGI2/PGE1-induced inhibition of TXA2 and PDGF release and thrombin generation (Table 4).
The effect of calcium channel blockers on thrombin generation and PDGF release could also be demonstrated in vivo in SCI subjects taking oral calcium channel blockers such as verapamil or nifedipine. The relative rate of thrombin generation and PDGF levels in the SCI subjects (n = 6) not prescribed calcium channel blockers was 2.2 ± 0.2 and 21.31 ± 3.2 pg/106 cells, respectively. However, after administration of the calcium channel blockers in these same subjects, the levels of thrombin generation and PDGF release were normalized to 1.09 ± 02 and 6.7 ± 0.3 pg/106 cells, respectively (Table4). Calcium channel blockers in the presence of PGI2 strongly inhibited PDGF release and thrombin generation.
Platelets in individuals with SCI were neither hypersensitive to aggregating agonists nor resistant to the inhibitory effect of PGI2/PGE1on platelet aggregation. However, platelet-stimulated thrombin generation and PDGF release from SCI platelets were not inhibited by the PGI2-stimulated increase in cAMP formation. The effect of PGI2/PGE1on platelets has been shown to be mediated through the binding of the agonist to its specific receptors on the cell surface (8, 21, 36). The binding of the prostanoid to its receptors activates the membrane-bound adenylate cyclase, increasing cellular cAMP and leading to the inhibition of platelet function. PGI2 and PGE1 bind to the same receptors on platelets, as well as to the purified receptor from platelets (8). Scatchard analysis has established the existence of high- and low-affinity PGI2/PGE1receptors on platelets. The binding of the prostanoid to the low-affinity (K din μM range) receptor is generally believed to increase cAMP level in platelets, which inhibits platelet aggregation (24). The binding of the prostanoid to the high-affinity (K d in nM range) receptors also increases the cAMP level but probably in a compartmentalized manner and in smaller quantity than low-affinity binding. The increase of cAMP through high-affinity prostanoid binding is physiologically more important than that of low-affinity binding because the plasma level of PGI2, even when stimulated, is in the 3–15 nM range, which corresponds to the K d of the high-affinity receptors. The synthesis of cAMP as a result of high-affinity PGI2 binding is under the feedback inhibition of the nucleotide itself (24). Also, the PGI2 binding to its high-affinity, but not to the low-affinity, receptors is inhibited by GTP (27). However, the data presented herein indicate that inhibition of platelet aggregation by increasing the cAMP level through the low-affinity prostanoid receptor binding exclusively did not result in the inhibition of either PDGF release or platelet-stimulated thrombin generation. This lack of inhibitory effect of PGI2 was not related to either the inhibition of platelet aggregation or cAMP formation by the prostanoid, but it appears to be related to the loss of platelet high-affinity PGI2 receptors.
Furthermore, the data herein indicate that impairment of the PGI2 effects in SCI platelets was the consequence of the decrease of high-affinity PGI2 receptors on these platelets. This can be supported by several lines of evidence.1) Treatment of non-SCI platelets with the purified IgG from the plasma of chronic SCI subjects, which specifically blocked the binding of PGI2 to its high-affinity receptor sites on these platelets, resulted in the impairment of PGI2 effects both in the release of PDGF and in platelet-stimulated thrombin generation in these platelets (23). 2) Restoration by insulin of the decreased high-affinity PGI2 receptors to normal ranges on the platelets from SCI subjects also effectively restored these PGI2 effects.3) Treatment of SCI platelets with Ca2+ channel blockers, like insulin, not only “corrected” the efficacy of PGI2 receptors in the inhibition of PDGF release and thrombin generation in these platelets but also simultaneously upregulated the high-affinity PGI2 receptors. Thus the up- or downregulation of high-affinity PGI2 receptors on the platelet surface resulted in the increased or decreased PGI2 effects in platelet function. Furthermore, the data herein indicated that it was the decrease of high-affinity PGI2 receptors on the platelet surface in SCI subjects and not the efficacy of these cells in the synthesis of cAMP (through the binding of PGI2 to its low-affinity receptors on the platelets) that plays a critically important pathophysiological role in atherosclerosis and thrombosis in SCI. Additionally, the data demonstrated that the high-affinity PGI2 receptors on the platelet membrane bilayer are not merely for the availability of prostaglandin in the activation of adenylate cyclase through low-affinity binding of the agonist, as previously thought (27), but that the high-affinity PGI2 receptors play an extremely important role in platelet function in the genesis of CAD in SCI subjects.
However, it should be mentioned that the increase of cAMP in platelets is generally believed to be associated with both the inhibition of platelet aggregation and the release reaction (11). The data herein indicated that increase of cAMP in platelets through the low-affinity PGI2-receptor binding in SCI was not sufficient for inhibition of the release reaction and that simultaneous binding of the protanoid to its high-affinity PGI2 receptors on platelets was essential for the PGI2 effect in the PDGF release reaction. However, the possibility exists that a cyclic independent pathway may be responsible for the release reaction or that the production of additional effector molecules may be required for the inhibition of the release reaction produced through high-affinity PGI2-receptor binding in the membrane bilayer through Ca2+ channel blocking. Uncoupling of platelet aggregation and the release reaction in the absence of cAMP synthesis is known (38). The upregulation of PGI2-receptor number on the platelet membrane by insulin has been recently established. Because platelets do not synthesize protein and PGI2 receptors are protein macromolecules (8), the increase of PGI2 receptors on the platelet surface by insulin (23) must be related to the translocation of these molecules from the “spare receptors” pool in the membrane bilayer to the outer membrane surface. It has been demonstrated that the increased availability of these spare receptors by insulin is related to the ADP-ribosylation of the Giα subunit by insulin, which may also be related to Ca2+channel blocking (15).
It could be argued that SCI subjects may have been prescribed various classes of medications, and as such the possibility exists that loss of high-affinity PGI2/PGE1binding may be due to receptor-binding interference by commonly used medications. However, incubation of normal platelets with these routinely prescribed medications did not interfere significantly with the binding of [3H]PGE1(20).
Increased levels of platelet-stimulated thrombin and PDGF release would be expected to have significant consequences on the pathogenesis of premature atherosclerosis in individuals with SCI. Thrombin and PDGF are potent mitogenic agents for arterial smooth muscle cells and induce the proliferation of human mesengial cells in the vascular intima (7). Thrombin is both a powerful aggregating agent and an important proteinase that is capable of converting fibrinogen to fibrin. Thus thrombin and PDGF not only have an essential role in blood coagulation but are believed to have a significant influence in the development of CAD and in the progression of renal failure. Although the effects of thrombin and PDGF are counteracted by several inhibitors present in plasma (5), these effects are, in part, inhibited by PGI2, a platelet-mediated interaction (3, 5, 11). The PGI2-induced increase in platelet cAMP not only inhibits platelet aggregation and the release reaction, of which PDGF is one constituent, it also inhibits platelet-stimulated thrombin generation (3, 12, 18). Thus, through its multi-inhibitory effects, PGI2 exerts a significant beneficial effect in the prevention of atherosclerosis (16, 17, 21, 31,39, 40). However, the findings herein indicate that not all the platelet-mediated effects of PGI2are through platelet cAMP levels. The failure of PGI2 to inhibit PDGF release and platelet-stimulated thrombin generation in SCI platelets was due to the loss of high-affinity PGI2receptors on the platelet surface. Restoration of these receptors would be expected to be beneficial. Because insulin has been shown to increase both the high- and low-affinity PGI2-receptor numbers in platelets without an effect on receptor affinities (21, 23) and because PDGF-stimulated cells require insulin to grow (25), treatment of SCI platelets with insulin increased high-affinity PGI2-receptor numbers sufficiently to “correct” PDGF release and thrombin generation to within the normal range.
The second messenger role of cAMP in the inhibition of platelet function has been demonstrated to be controlled by the countervailing effect of cytosolic Ca2+ ion, which has been shown to be coupled to various intracellular messenger functions in the activation of platelet function (31). A cytosolic elevation of Ca2+ ions, due to increased influx or intracellular mobilization, results in the agonist-induced activation of platelets and the release reaction (9). The inhibition of platelet activation by the intracellular increase of cAMP by prostaglandins is mediated through the extrusion of cytosolic Ca2+ ions through a Ca2+-Mg2+-ATPase system in the plasma membrane (14, 31) and inhibition of Ca2+ fluxes. However, in SCI individuals, either in vivo or in vitro treatment with calcium channel blockers was able to reverse the abnormal PDGF release and normalize PGI2/PGE1platelet-stimulated thrombin generation.
Furthermore, the upregulation of PGI2-receptor numbers by insulin or the blocking of Ca2+ channels by different Ca2+ channel blockers resulted in the normalization of release of both PDGF and TXA2 (Table 4). Because these release-reaction products are synthesized by two entirely different pathways and PDGF, unlike TXA2, is not synthesized in platelets, the inhibition of release of these substances in SCI platelets in the presence of PGI2 + insulin and by the Ca2+ channel blockers was the consequence of the inhibition of the platelet-release reaction and not due to the inhibition of their synthesis in platelets. Because the blockade of the high-affinity PGI2-receptor antibody resulted in the impairment of these effects of PGI2 in normal platelets (18), it could be concluded that these effects of PGI2 were mediated through the blocking of Ca2+ channels through the interaction of PGI2 with its high-affinity receptors on the platelet surface.
The failure of PGI2 to inhibit the release reaction in SCI platelets when compared with normal platelets, even though the inhibition of platelet aggregation induced by the prostanoid was similar in both the cases, supported the earlier report (38) that aggregation of platelets and the release reaction are not always coupled, in that one of these events may occur in the absence of the other and probably involves Ca2+ mobilization in platelets (Tables 1 and 4).
The mechanism by which PGI2, through high-affinity binding, inhibits PDGF release and thrombin generation is unknown but could be due to the blocking of Ca2+ channels in platelets mediated through the binding of the prostanoid to its high-affinity receptor sites on the cell surface. No method is currently available that permits differentiation between the cellular activities of high- and low-affinity PGI2/PGE1receptors. The inhibitory effect of calcium channel blockers may be speculated to be mediated through the increase of cAMP via the high-affinity PGI2/PGE1-receptor interaction and inhibition of agonist-induced Ca2+ influx. Calcium antagonists have been shown to stimulate PGI2production and to inhibit platelet aggregation and thrombus formation (26, 30). Insulin also attenuates agonist-induced Ca2+ influxes (34). The use of Ca2+ channel blockers, such as verapamil or nifedipine, may be beneficial in individuals with SCI by the correction of PDGF release and inhibition of thrombin generation, thus preventing or attenuating the deleterious effect on the vasculature.
Sincere thanks to Dr. Sinha for constructive comments in preparation of this manuscript.
Address for reprint requests and other correspondence: N. Kahn, Spinal Cord Damage Research Center, Veterans Affairs Medical Center, 130 West Kingsbridge Rd., Room 1E-02, Bronx, NY 10468 (E-mail:).
This work was supported by the Eastern Paralyzed Veterans Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 1999 the American Physiological Society