Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIbα

Abstract

SummaryWe studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIbα-von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a coneand-plate viscometer (shear: 5,000 s−1 for5 min) in the presence of antagonists to GPIbα (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared fromACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIbα with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ∼30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIbα, explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIbα with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.

Laser-induced primary and secondary hemostasis dynamics and mechanisms in relation to selective photothermolysis of port wine stains

“…Although the major physiological function of CD42b is the adhesion of circulating platelets to vWF in the subendothelial matrix at high shear, which leads to activation of integrin a IIb b III (GPIIb/IIIa) and subsequent aggregation [26], it has also been shown to mediate platelet adhesion under low shear conditions, i.e., in venules [27,28]. Inhibition of CD42b has further been correlated to significantly reduced platelet microparticle formation [29]. At a shear rate of 7.0 AE 3.7 s À1 in hamster venules, CD42b may therefore constitute an important receptor during the hemodynamic response, inasmuch as both platelet aggregation and coagulation prevail.…”

Biological effects of dynamic shear stress in cardiovascular pathologies and devices

“…The effect was more pronounced at shear stress higher than 10 dynes/cm 2 , which confirmed the existence of threshold for SIPA [67,108]. Platelet activation, aggregation and microparticle release, either alone or with RBC, on exposure to normal or pathological shear stress have since been extensively investigated in cone and plate or Couette viscometers [46,51,52,[109][110][111][112]. However, these studies did not consider diverse flow dynamics and their effects on SIPA such as stagnation, flow reversal, local turbulence and eddy currents, as might be expected in vivo when platelets pass through complex geometries.…”

Platelet receptor gain-of-function single nucleotide polymorphisms in carotid and vertebral stenosis patients

“…This receptor and its interaction with vWF is of particular interest, because it is functional at the high shear rates found in arterial stenosis. Its inhibition results in reduced generation of platelet derived microparticles, thrombin generation and procoagulant activity at high shear forces [17,18]. Furthermore, in vitro studies have shown that platelet membrane glycoprotein polymorphisms (including the Kozak sequence) influence experimental thrombus formation at typical shear rates [30].…”

“…The GPIba (5T/C) SNP was of particular interest, since the A1 binding site of von Willebrand factor (vWF) may be activated to bind platelet GPIba under high shear stress, as it occurs in stenotic atherosclerotic lesions [16][17][18][19]. So far, clinical studies investigating the implications of the GPIba (5T/C) polymorphism have yielded controversial results [4][5][6][7]20].…”