Greater inhibition of platelet procoagulant activity by antibody‐derived glycoprotein IIb–IIIa inhibitors than by peptide and peptidomimetic inhibitors

Abstract

Summary. Antibody-derived GPIIb±IIIa antagonists, such as the c7E3 Fab fragment abciximab, have been shown to inhibit platelet procoagulant activity as well as platelet aggregation. Whether low-molecular-weight peptide-derived and peptidomimetic antagonists also inhibit platelet procoagulant activity in a similar manner has not been fully investigated. We compared the effects of the antibodyderived antagonists c7E3 Fab and m10E5 IgG, the peptidederived antagonists eptifibatide, MK-852 and RGDS, and the peptidomimetic RO44±9883 on platelet procoagulant activity and on the stimulated cytosolic calcium increases that promote procoagulant activity. Procoagulant activity was measured as prothrombinase activity in gel-filtered platelets, activated by collagen plus thrombin or collagen alone, with and without stirring. The stimulated increases in cytosolic calcium were measured in parallel samples of platelets loaded with fura-2AM. Both c7E3 and m10E5 inhibited prothrombinase activity by 40±50% under all conditions of activation tested and inhibited cytosolic calcium increases to a similar extent in stirred, but not unstirred, platelets. The low-molecular-weight antagonists caused significantly less inhibition of prothrombinase activity in collagen plus thrombin-stimulated platelets, and produced no inhibition but rather a slight enhancement of activity in platelets stimulated by collagen alone. These antagonists also had little or no effect on the cytosolic calcium increases in stirred platelets. These differential effects of antibody-derived versus non-antibody GPIIb±IIIa antagonists on procoagulant activity may be a factor contributing to the differing anti-thrombotic effects of these antagonists seen in clinical trials.

Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation

“…However, the measurements mostly concerned initial Ca 2+ rises, while in our hands only late Ca 2+ signals appear to be affected. Interestingly, one study does describe long-term inhibition of collagen/thrombin-induced Ca 2+ responses with abciximab but not with other integrin blockers specifically under conditions of stirring [23]. This contrasts with the present findings where appropriate concentrations of different integrin blockers all had similar effects.…”

“…Several groups reported that integrin blockers were unable to change platelet Ca 2+ responses to collagen and/or thrombin [22, 23, 39]. However, the measurements mostly concerned initial Ca 2+ rises, while in our hands only late Ca 2+ signals appear to be affected.…”

“…Early reports suggest that various integrin α IIb β 3 antagonists differently affect thrombin generation in platelet-rich plasma (PRP) [19, 23]. To verify this, we prepared human PRP and determined the effects on tissue factor-induced thrombin generation of three α IIb β 3 blockers, all in clinical use: the human/mouse chimeric monoclonal antibody fragment abciximab (blocking integrins α IIb β 3 and α v β 3 ), the peptide mimetic eptifibatide, and the non-peptide sulfonamido compound, tirofiban.…”

“…Suggestions are that the integrin is (1) involved in the formation of procoagulant microparticles [17–19], (2) directly binds prothrombin [20], or (3) provides binding sites for factor Va and other coagulation factors [21, 22]. Studies so far are complicated by data that show that distinct α IIb β 3 antagonists may differ in their effects on platelet activation and procoagulant activity [19, 23]. Another complicating factor is that part of the role of α IIb β 3 may be secondary to that of autocrine ADP, which enhances PS exposure via P2Y 12 receptor stimulation [24–26].…”

The role of platelets in blood coagulation—effects of platelet agonists and GPIIb/IIIa inhibitors studied by free oscillation rheometry

“…This is in accordance with earlier reports about an increased procoagulant activity in platelets stimulated with collagen and thrombin in the presence of MK-852, but not with thrombin as single stimulus [20].…”