Pharmacodynamic characterization of the interaction between the glycoprotein IIb/IIIa inhibitor YM337 and unfractionated heparin and aspirin in humans

Abstract

Aims

To investigate the pharmacodynamic interaction of unfractionated heparin (UFH) and acetylic salicylic acid (ASA) on YM337, a monoclonal humanized antibody of the platelet GPIIb/IIIa receptor.

Methods

In a randomized, placebo-controlled study three treatment groups each with six healthy volunteers received the following medication: group 1, ASA (3 days) + UFH + YM337 (placebo); group 2, ASA (placebo) + UFH (placebo) + YM337; group 3, ASA + UFH + YM337. Assessments were made over 24 h and included bleeding time (BT), ADP (20 µm)- and collagen (5 µg ml⁻¹)-induced platelet aggregation and PAC1 and CD62 expression measured by flow cytometry.

Results

In group 3 BT was prolonged to 35 [median, 16–45 min (1,3 quartile)] after UFH administration, increasing to 45 [median, 42–45 min (1,3 quartile)] after YM infusion (6 h). BT remained elevated to 26 [median, 14–45 min (1,3 quartile)] at 24 h, while groups 1 and 2 returned to normal values. Collagen-induced aggregation was 73% [median, 70–80% (1,3 quartile)] under YM337 alone, 79% [median, 72–80% (1,3 quartile)] under ASA + UFH and reduced only in group 3 to 24% [median, 18–29% (1,3 quartile)]. In both groups receiving active YM337, PAC1 expression showed a reduction to <20% after 6 h of infusion. CD62 expression was not significantly affected by any treatment.

Conclusion

UFH and YM337 have strong synergistic effects on BT, while coadministration of ASA strongly augments inhibitory effects of YM337 on collagen-induced platelet aggregation.

Introduction

The clinical benefit of the intravenous GPIIb/IIIa receptor antagonists is proven only with an underlying comedication of unfractionated heparin (UFH) and aspirin [1–3]. Although heparin and aspirin seem to be a pivotal component of the antithrombotic regimen with GPIIb/IIIa inhibitors, these drugs also contribute to the bleeding risks of this therapy [3]. It has been shown for several GPIIb/IIIa inhibitors that due to their potent inhibitory effects on platelets, the formation of thrombin on the platelet membrane is considerably decreased [4–6], possibly reinforcing the bleeding risk under heparin. UFH can provoke platelet activation in vitro and in vivo [7, 8], which may counteract the anti-aggregatory effects of antiplatelet drugs. GPIIb/IIIa inhibitors have strong inhibitory effects on ADP- or TRAP-induced aggregation, when given alone. Both agonists are commonly used in clinical trials with GPIIb/IIIa inhibitors investigating platelet aggregation [9–11]. Collagen-induced aggregation on the other hand has rarely been used in the pharmacodynamic assessment of GPIIb/IIIa inhibitors, usually in phase I studies [5, 12] and in lower concentrations (1–2 µg ml⁻¹). It is a common test to detect aspirin effects on platelets [13]. In vitro data suggest that combined treatment with aspirin and a GPIIb/IIIa inhibitor might have synergistic effects on collagen-induced aggregation, whereas effects of GPII/IIIa inhibitors alone on collagen-induced aggregation are less pronounced [14, 15]. The pharmacodynamic profile of the interaction between GPIIb/IIIa inhibitors and aspirin and heparin might therefore be of interest. We investigated UFH given together with YM337, a humanized monoclonal GPIIb/IIIa inhibitor [5], on an underlying aspirin medication, resembling a common treatment strategy in acute coronary syndromes, and compared these data to those under the GPIIb/IIIa inhibitor alone and to UFH with aspirin alone. In a first human study, regarding the extent and rate of recovery of platelet inhibition, the pharmacodynamic profile of YM337 was similar to that of abciximab [5].

Methods

The study was conducted in a randomized and placebo-controlled, parallel group design. All laboratory personnel evaluating the pharmacodynamic parameters were blinded for the treatment allocation. After approval by the local Institutional Review Board and obtaining informed consent, 18 healthy male volunteers (age 18–40 years, weight 52–98 kg) were examined. None of the volunteers had taken drugs or medications during the last 14 days before the study. Study subjects were randomized to three treatment groups: (1) acetylic salicylic acid (ASA) + heparin + placebo (n = 6); (2) placebo + placebo + YM337 (n = 6); and (3) ASA + heparin + YM337 (n = 6). Each volunteer received ASA (325 mg) or matching placebo for 3 days. At day 3 after ASA intake, at −1 h, subjects of treatment groups 1 and 3 received a bolus of UFH of 70 IU kg⁻¹ (maximum 5000 IU) followed by a weight-adjusted infusion of 15 IU kg⁻¹ h⁻¹ (maximum 1000 IU h⁻¹) for 7 h, group 2 received matching placebo. One hour after drug application (ASA and UFH or matching placebo) at day 3, YM337 was administered intravenously as bolus and infusion of 0.25 mg kg ⁻¹+ 1.0 µg kg⁻¹ min⁻¹ for 6 h.

Bleeding time

Bleeding time (BT) was measured at day 1 (0 h) and at day 3 (−1 h, 0 h, 1 h, 6 h and 24 h). For the measurement of bleeding time a sphygmomanometer cuff was inflated to 40 mmHg in the upper arm. A standardized horizontal laceration was created on the volar side of the forearm by using Simplate devices (Organon Teknika, Eppelheim, Germany). Blood was wiped from the cut with filter paper after 15 s and then at 15-s intervals until the bleeding had stopped (normal range 4–10 min).

Flow cytometric assessment of expression PAC1 and CD62

Blood samples were collected into sodium citrate (3.13%) tubes at day 1 (0 h) and day 3 (−1 h, 0 h, 15 min, 3 h, 6 h, 12 h and 24 h). For platelet activation, TRAP (H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-OH) was added at a final concentration of 10 µm. Antibodies (Ab) used were anti-CD62 Ab (CD62–FITC, IgG1 mouse; Cymbus Biotechnology, Hampshire, UK), PE-conjugated anti-CD42b Ab (CD42b–PE, IgG1 mouse; Coulter-Immunotech, Paris, France) and FITC-conjugated anti-PAC1 Ab (FITC–anti-PAC1, IgG1 mouse). PAC1, a murine IgG-κ mAb, binds to a conformation-dependent determinant on the activated GPIIb–IIIa complex. Flow cytometric measurement was carried out using a FACScan ™ flow cytometer (Becton Dickinson, Heidelberg, Germany). To establish resolution for acceptance of platelet events only, binding of PE-labelled platelet-specific anti-CD42b was used to set a gate. Difference of fluorescence intensity at day 1 was set to 100% and results were related to this maximal value and expressed in percent. Nonspecific membrane immunofluorescence was determined by the isotype-matched FITC-conjugated IgG control. Platelets positive for CD62 or PAC1 were defined as those events in the electronic gate which had a fluorescence intensity greater than a threshold set at 1% when the same cells were labelled with the isotype control. CD62 expression was quantified as percentage of CD62-stained platelets of the gated population. PAC1 expression, since all platelets are positive, was presented as the mean fluorescence intensity (MFI given in arbitrary units) of the positive platelet population. Acquisition and processing of data from 5000 platelets were carried out with CONSORT software (Becton Dickinson).

Platelet aggregation

ADP- and collagen-induced platelet aggregation was assessed at day 1 (0 h) and day 3 (−1 h, 0 h, 15 min, 3 h, 6 h, 8 h, 10 h, 12 h, and 24 h). Platelet-rich plasma (PRP) was prepared by centrifugation of citrated blood at 400 g for 3 min at 24 °C and the PRP was removed. Platelet aggregation was assessed in PRP using a turbidimetric light-transmittance device (APACT Labor, Ahrensburg, Germany) within 1 h after collection. Inducers were adenosindiphosphate (ADP; DADE-Behring, Heidelberg, Germany) at a final concentration of 20 µm as well as collagen (SKF Horm; Nycomed, Ismaning, Germany) at 5 µg ml⁻¹ final concentration The aggregation response is given as percentage of maximal light transmission A_max.

Activated partial thromboplastin time and prothrombin time

Blood sampling for analysis was done at day 1 (0 h) and at day 3 (−1 h, 0 h, 15 min, 30 min, 3 h, 6 h, 8 h, 10 h, 12 h and 24 h). After collection blood samples were immediately centrifuged at 2000 g for 10 min, and activated partial thromboplastin time (aPTT) and prothrombin time (PT) were determined by an automated device (ACL 7000, Ahrensburg, Germany).

Statistical evaluation

Data were described by use of median box plots (median, 1 quartile, 3 quartile, range) in figures and by median and quartiles in tables. For each time point of the sampling schedule, pharmacodynamic parameters (fibrinogen binding, CD62, platelet aggregation, bleeding time) were analysed for differences between the study groups by analysis of variance according to Kruskal–Wallis, adjusted for multiple comparisons (group 1 vs. 2, 2 vs. 3 and 1 vs. 3) according to Holms. All graphics were generated using SPSS (Chicago, IL, USA).

Results

The study medication was well tolerated and no side-effects (minor or major bleeding, thrombocytopenia) occurred during the administration.

Bleeding time

BT was not prolonged under ASA treatment alone (day 3, −1 h). Coadministration of UFH (day 3, 0 h) led to a prolongation of BT to 27 min (median) (group 1) or 35 min (median) (group 3), respectively. Under YM337 alone (group 2), bleeding time was prolonged almost three-fold at bolus (median 29 min) and after 6 h of infusion (median 26 min), whereas in group 3 (combined treatment) bleeding time was significantly longer (median 45 min at bolus and after 6 h) compared with the other treatment groups. At 24 h bleeding time remained elevated to 26 min (median) in group 3, whereas groups 1 and 2 returned to normal values (Figure 1).

Figure 1.

Bleeding time (median box plots, n = 6 for each bar). Acetylic salicylic acid (ASA) + unfractionated heparin (UFH), YM337, □ ASA + UFH + YM337. *P < 0.02 from corresponding value of treatment with YM337 alone.

Expression of CD62 and PAC1

In both groups receiving active YM337, PAC1 expression showed a strong reduction after the bolus to 10% (median) (group 2) and 9% (median) (group 3), and remained reduced to <20% after 6 h of infusion. Recovery of PAC1 at 12 h was more pronounced with YM337 alone (median 65%) compared with combination with ASA and UFH (median 26%, P < 0.02), but similar at 24 h. CD62 expression was not significantly affected by any treatment (Table 1).

Table 1.

Flow cytometric parameters: median (25 and 75 quartile).

		Time
	Treatment group	Day3 (–1 h)	0 h	0,25 h	6 h	12 h	24 h
Parameter	actual treatment*	Under ASA	under ASA + UFH		under ASA + UFH + YM337
PAC1-expression (% of pretreatment at day 1, 0 h)	1 ASA + UFH	95 (86–112)	104 (88–121)	94 (87–115)	91 (86–107)	88 (84–110)	90 (71–119)
	2 YM337	107 (105–115)	103 (101–111)	10 (4–18)	5 (2–11)	65 (40–89)	67 (47–83)
	3 ASA + UFH + YM337	111 (99–137)	103 (90–122)	9 (2–19)	2 (1–3)	26 (22–45)†	60 (50–86)
CD62-expression (% of pretreatment at day 1, 0 h)	1 ASA + UFH	103 (93–120)	103 (99–107)	107 (95–113)	106 (99–114)	101 (90–125)	94 (76–121)
	2 YM337	113 (92–117)	122 (101–133)	99 (86–126)	104 (87–118)	116 (93–128)	113 (98–132)
	3 ASA + UFH + YM337	103 (93–120)	106 (94–120)	86 (71–103)	84 (76–103)	91 (77–108)	82 (71–95)
		13 (92–117)
		108 (100–131)

^*refers to actual treatment with an active drug or the corresponding placebo.

^†P < 0.02 vs group 2.

Platelet aggregation

Platelet aggregation induced by 20 µm ADP was inhibited to approximately 20% during the infusion period in all groups receiving active YM337. Inhibition of aggregation recovered fast in both group 1 and group 3 after the end of infusion (Figure 2). ASA treatment did not inhibit aggregation induced by 5 µg ml⁻¹ collagen. YM337 alone only marginally affected aggregation to 5 µg ml⁻¹ collagen from 82% (median) before the bolus to 73% (median) at 6 h. However, combination of ASA + UFH + YM337 reduced collagen-induced aggregation to 24% (median) after bolus and at 6 h with a rapid recovery to 49% at 12 h (Figure 3).

Figure 2.

Aggregation response (median box plots, n = 6 for each bar) to 20 µm adenosindiphosphate (ADP). acetylic salicylic acid (ASA) + unfractionated heparin (UFH), YM337, □ ASA + UFH + YM337, P < 0.02 from corresponding value of treatment with YM337 alone.

Figure 3.

Aggregation response (median box plots, n = 6 for each bar) to 5 µg ml⁻¹ collagen. acetylic salicylic acid (ASA) + unfractionated heparin (UFH), YM337, □ ASA + UFH + YM337. *P < 0.02 from corresponding value of treatment with YM337 alone.

aPTT and PT

In both groups under active UFH treatment, aPTT reached the peak immediately after the bolus to >500 s, at 6 h aPTT was prolonged up to three-fold to approximately 140 s. No differences were seen between groups treated with active UFH, and a rapid recovery to normal values was observed at 8 h, i.e. 2 h after cessation of the infusion. PT time was slightly prolonged by 1.2-fold after the UFH bolus and at 6 h under UFH infusion.

Discussion

Key findings of this interaction study were: (i) compared with ASA alone, coadministration of UFH significantly enhanced bleeding time even before application of the GPIIb/IIIa inhibitor YM337, and strongly reinforced the effects of the GPIIb/IIIa inhibitor on bleeding time; (ii) aspirin augments the inhibitory effects of the GPIIb/IIIa antagonist on collagen-induced aggregation in a supra-additive manner; and (iii) expression of CD62, a marker of platelet secretory function, was hardly reduced by any treatment. Our results suggest that aspirin has strong synergistic effects on GPIIb/IIIa inhibitors regarding inhibition of collagen-induced aggregation. Although the study was carried out on healthy subjects, all volunteers underwent a clinically relevant dosing scheme of the study drugs.

Experimental studies with different sources and concentrations of heparin and different agonists have variously documented platelet activation [7, 16] or platelet inhibition [8, 17]. The pro-aggregatory response was detected with various agonists (ADP, collagen, adrenaline and TRAP), more consistently with low concentrations of ADP and less consistently with higher ADP concentrations [18]. Therefore it was suggested that the pro-aggregant effect of UFH is modest and can be overcome with strong platelet inhibition [7]. A recent publication showed that therapeutic heparin concentrations augment platelet inhibition by abciximab in vitro [8]. We might have failed to detect any activating effects of UFH in our study since we administered heparin only with an underlying ASA co-medication, which might have prevented platelet activation. On the other hand, even after cessation of UFH and a decline of aPTT at 12 h and 24 h, the effects of GPIIb/IIIa inhibition on bleeding time are sustained, presumably by small amounts of circulating or vessel wall-bound UFH [19].

Collagen-induced aggregation has only rarely been used for the demonstration of clinical platelet inhibition. Most clinical studies with abciximab and integrilin employed 20 µm ADP [20, 21], and in studies with tirofiban even 5 µm ADP were used for aggregometry [22]. The choice and concentration of the agonist have an impact on the platelet activation achieved and the ability to detect inhibition of certain pathways. When given alone, inhibitory effects of GPIIb/IIIa inhibitors on collagen-induced aggregation seem to be smaller than effects on ADP-induced aggregation in vitro [14], and in vivo [5, 23]. Compared with inhibition of 20 µm ADP-induced aggregation, effects of YM337 alone on 5 µg ml⁻¹ collagen-induced aggregation are only small. Some reports quoting stronger in vivo effects of GPIIb/IIIa inhibitors on collagen-induced aggregation [12, 24] used lower concentrations of collagen (2 µg ml⁻¹), which were even inhibited by the intake of 300 mg day⁻¹ aspirin alone [14]. This is the first study which proves the in vivo interaction between aspirin and GPIIb/IIIa inhibition, although several in vitro investigations have been done. The aggregation response to collagen reflects an important step in the initiation of thrombus formation, since collagen is a pivotal component of the subendothelial matrix [25, 26]. Platelets express a variety of collagen receptors [27] and besides integrin α₂β₁, it has widely been accepted that the GPVI/FCRγ-chain is required for collagen-induced platelet aggregation. Since the receptors for platelet activators like ADP or thromboxane A₂ (TXA₂) couple to several heterotrimeric G proteins, an interaction is likely, as recent studies with Gαq-deficient mice indicate [28], but the exact interactions are poorly understood at the moment. GPVI activation with collagen, on the other hand, is involved in inside-out signalling of platelets leading to GPIIb/IIIa activation [29], and it has been supposed that the generation of TXA₂ enhances the expression of activated GPIIb/IIIa complexes. However, we suppose that the underlying comedication with ASA, although it does not lead to an independent effect on the strong collagen stimulus (5 µg ml⁻¹), might exert a synergistic effect on the inhibition of the GPIIb/IIIa receptor by YM337, since the physical principle of measuring the aggregation response (enhanced light transmittance after aggregation of PRP) is based on the GPIIb/IIIa–fibrinogen interaction. This assumption is also supported by an in vitro investigation from our group [14], showing that GPIIb/IIIa inhibitors had only a minor effect on collagen-induced aggregation in native blood from healthy subjects, whereas the same concentration of the IIb/IIIa inhibitors completely inhibited collagen-induced aggregation when the subjects were pretreated with aspirin. Similar effects were presented in a previous in vitro investigation [30].

Platelet granular secretion occurs with platelet activation and aggregation and α-granular proteins like platelet derived growth factor may have sustained effects on vascular wall remodelling [31]. Release of α-granule contents are associated with the appearance of CD62 (P-selectin), which acts as ligand and mediates the adherence of degranulated platelets to leucocytes, initiating inflammatory responses [32, 33]. CD62 expression is enhanced in patients with acute coronary syndromes [34] and has been described as predictor of restenosis after percutanerous transluminal coronary angioplasty [35]. In this study pretreatment with aspirin alone had no influence on CD62 expression, consistent with recent investigations by our group [14] and others [35, 36], and CD62 expression was not affected by the infusion of the GPIIb/IIIa inhibitor YM337.

In conclusion, our results show strong synergistic effects of the combination of YM337 with ASA and UFH on the prolongation bleeding time and inhibition of collagen-induced platelet aggregation.