Effect of Calcium Channel Blockers on Platelet GPIIb-IIIa as a Calcium Channel in Liposomes: Comparison with Effects on the Intact Platelet

Abstract

SummaryThe platelet membrane glycoprotein IIb-IIIa complex is essential for platelet aggregation and functions as a fibrinogen receptor on the activated platelet. When incorporated into phospholipid vesicles, this glycoprotein complex can function as an apparent calcium channel which facilitates the transit of calcium across a phospholipid barrier. In order to further evaluate this calcium channel, the effect of calcium channel blockers of the dihydropyridine (nifedipine and nicardipine), arylalkylamine (verapamil) and benzothiazipine (diltiazem) classes were evaluated on GPIIb-Illa liposomes with encapsulated fura-2 (a fluorescent calcium indicator). Nicardipine, verapamil, and nifedipine significantly inhibited calcium influx into GPIIb-IIIa liposomes; however, this required 190 μM, 400 μM, and 140 μM drug, respectively. These concentrations are 10-1,000 fold greater than those clinically obtainable. In contrast, diltiazem at concentrations >220 μM and amiloride at concentrations >800 μM showed no inhibitory effects. When aspirinized platelets were activated with 30 μg/ml bovine fibrillar collagen, both nicardipine and diltiazem produced a decrease in both the initial rise and maximum cytoplasmic calcium concentration. Parallel experiments were performed to assess the effects of verapamil, nicardipine, and diltiazem on platelet aggregation in platelet rich plasma. Nicardipine, 190-380 μM, induced a prolongation of the lag phase, but no effect on the final degree of platelet aggregation to collagen. Similar inhibition of platelet aggregation was seen with diltiazem and verapamil although the effect of diltiazem was less pronounced particularly at higher concentrations of collagen. No effect was seen on aggregation with 32 μM ADP which is release independent, or on the primary wave of low dose ADP induced platelet aggregation. Verapamil (200 μM) and nicardipine (>190 μM) partially inhibited the secondary wave of 3.2 μM ADP aggregation. In contrast, diltiazem (400 μM) had limited effect on ADP induced aggregation. Similarly, both nicardipine and verapamil markedly inhibited platelet aggregation to 6 μM epinephrine; however, diltiazem had no effect. These data demonstrate that the GPIIb-IIIa calcium channel is selectively inhibited by different classes of calcium channel blockers. This inhibition requires concentrations much higher than that to inhibit classic voltage gated calcium channels and may be by a different mechanism. Further evaluation will determine whether this differential inhibition of the GPIIb-IIIa calcium channel is related to differences observed in platelet aggregation.

Effects of diltiazem on platelet activation and cytosolic calcium during percutaneous transluminal coronary angioplasty

“…22 23 It has been proposed that calcium antagonists may exert their effects on platelet [Ca 2+ ] i by inhibition of calcium mobilization from intracellular stores, inhibition of GP IIb/IIIa complex, or interaction with receptor operated calcium channels such as α-adrenergic receptors. 24 Platelet activation after PTCA is generally associated with an adverse prognosis and is one of the primary targets of medication. We demonstrated that platelet activation that continued in the presence of conventional aspirin/ticlopidine treatment was inhibited by combined diltiazem and aspirin/ ticlopidine treatment.…”

Platelet morphology and plasma indices of platelet activation in essential hypertension: effects of amlodipine-based antihypertensive therapy

“…Platelet cytosolic calcium is involved in several aspects of platelet activation such as shape change, secretion and aggregation. Cytosolic calcium increases during platelet activation both as a result of release from intracellular stores, like the dense tubular system, and via in¯ux from the extracellular space (25,26). However, in contrast to smooth muscle cells or cardiac myocytes, platelets lack voltage-dependent calcium channels of the Ltype; the in¯ux of Ca 2 during platelet activation occurs through receptor operated calcium channels and through channels activated by the GpIIb/IIIa receptors (27,28).…”

Calcium channel antagonists in the modern era of coronary thrombolysis: Benefit or detriment?

“…They also increase intracellular cAMP concentrations [24], antagonize calmodulin activation [25], reduce thromboxane A 2 synthesis [26], inhibit serotonin uptake [27][28][29], inhibit platelet activating factor, and neutralize the important platelet surface receptor, glycoprotein IIb/IIIa [30]. These observations are particularly interesting given that platelets do not have classic L (high threshold) or T (low threshold) calcium channels nor do they have specific binding sites for the antagonists themselves.…”

“…These observations are particularly interesting given that platelets do not have classic L (high threshold) or T (low threshold) calcium channels nor do they have specific binding sites for the antagonists themselves. It may be that other channels (non-voltage gated) are involved [30].…”