Induction of platelet Ca2+ influx and mobilization by a monoclonal antibody to CD9 antigen

Abstract

We found that a monoclonal antibody (MoAb) to CD9 antigen, PMA2, induced a rise in cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded platelets, and we examined whether this response was due to direct action of PMA2 on CD9 antigen. The rise in [Ca2+]i was dependent on the PMA2 concentration, irrespective of the presence or absence of extracellular Ca2+. The role of secreted adenosine diphosphate (ADP) and thromboxane in the [Ca2+]i response to PMA2 was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. Combined treatment with CP/CPK and aspirin abolished the rise in [Ca2+]i, although either CP/CPK or aspirin alone produced only partial inhibition. Inhibition of adenosine triphosphate (ATP) secretion and thromboxane B2 synthesis by an MoAb to the glycoprotein IIb-IIIa complex, PMA1, resulted in little [Ca2+]i response to PMA2. In contrast, thrombasthenic platelets, in which ATP secretion and thromboxane B2 synthesis were normal, showed a normal [Ca2+]i response. When PMA2 was added to CD9+ mononuclear cells, no rise in [Ca2+]i was observed. Thus, we conclude that binding of monoclonal immunoglobulin G molecules to the CD9 antigen raises [Ca2+]i through the effect of secreted ADP and thromboxane on platelets, and that CD9 antigen is not directly involved in induction of Ca2+ influx and mobilization.

Antibody cross-linking of human platelet P-selectin induces calcium entry by a mechanism dependent upon Fcγ receptor IIA

“…Figures 3B and 3C show that NiCl 2 , at 5 mM, is able almost to abolish the rise in cell calcium, demonstrating that the major part of the response in these cells is through calcium entry rather than release from intracellular stores. This is consistent with previous reports where it had already been shown that FcγRIIA was able to mediate calcium entry in platelets (33,39,40) and other cells (41). Kuroda, et al (1995) (33) had previously shown that calcium entry plays a central role in signaling by FcγRIIA, and that the influx was not likely to result from capacitative mechanisms.…”

Apoptosis signal‐regulating kinase 1 regulates immune‐mediated thrombocytopenia, thrombosis, and systemic shock

“…release, secretion, and aggregation. 40,42,43,71 These findings suggest F I G U R E 6 Ask1 regulates thrombosis in a mouse model of ITT. (A) WT, hFcR/Ask1 +/+ , and hFcR/Ask1 -/mice were injected with anti-mGPIX-800CW (for in vivo labeling of platelets) followed by anti-mCD9 (or PBS vehicle control) to induce thrombosis.…”

“…Anti‐CD9‐induced Ca 2+ release and platelet aggregation are dependent on TxA 2 generation, notably at agonist concentrations near threshold 40‐43 . As Ask1 regulates TxA 2 generation by other ITAM‐receptors, 19 we next investigated ASK1’s role in regulating IC‐induced TxA 2 generation.…”

“…Indeed, TxA 2 generation is not absolutely required at higher doses because aspirin did not inhibit hFcR/Ask1 +/+ platelet aggregation or increase lag time at anti‐CD9 concentrations ≥1 ug/mL, but did so at lower concentrations 29 . This TxA 2 ‐independent mechanism also exists in human platelets because aspirin treatment did not completely block anti‐CD9‐induced Ca 2+ release, secretion, and aggregation 40,42,43,71 . These findings suggest that anti‐CD9 induces platelet activation via TxA 2 ‐dependent and TxA 2 ‐independent mechanisms, favoring TxA 2 ‐dependent mechanisms at low agonist doses.…”

“…Anti-CD9-induced Ca 2+ release and platelet aggregation are dependent on TxA 2 generation, notably at agonist concentrations near threshold. [40][41][42][43] As Ask1 regulates TxA 2 generation by other ITAM-receptors, 19 we next investigated ASK1's role in regulating TxA 2 generation, however, was delayed past 5 minutes in hFcR/ Ask1 -/platelets, only reaching a significant level 5 to 10 minutes after stimulation, corresponding to the start of hFcR/Ask1 -/aggregation. These data suggest that the increased lag-time of hFcR/ Ask1 -/platelets is related to delayed TxA 2 generation ( Figure 4A).…”