Effects of Anticoagulant on pH, Ionized Calcium Concentration, and Platelet Aggregation in Canine Platelet-Rich Plasma
In vitro platelet aggregation studies may be part of the diagnostic evaluation of a patient with suspected thrombopathia or the quality control of canine platelet transfusion products, such as apheresis platelet concentrates and cryopreserved platelets. While 3.8% sodium citrate (CIT) is the most commonly used anticoagulant for diagnostic studies, anticoagulant acid-citrate-dextrose (ACD-A) is the choice for collection of platelets by apheresis and for storage. The purpose of this study was to compare the effects of CIT and ACD-A, on pH, extracellular ionized calcium concentration [Ca2+], and platelet aggregation in canine platelet-rich plasma (PRP).
Blood was collected from 12 healthy mixed breed dogs into both CIT and ACD-A. The pH and [Ca2+] of PRP were measured by a NOVA, and platelet aggregation was assessed by optical aggregometry. Platelet agonists (final concentration) evaluated were ADP (20 µM), γ-thrombin (100 nM), and convulxin (20 nM). Washed platelets were used to evaluate the effects of pH and [Ca2+]. Amplitude (%) and slope of aggregation were recorded.
CIT PRP was alkaline (mean pH 7.56), whereas ACD-A PRP was acidic (mean pH 7.05). The [Ca2+] was significantly greater in CIT (mean, 0.224 mmol/L) than in ACD-A PRP (mean, 0.188 mmol/L). ADP-induced platelet aggregation was markedly diminished in ACD-A (mean, 3%) compared with CIT (mean, 54%). Platelets collected in ACD-A from all dogs responded to γ-thrombin with only platelet shape change in contrast to platelets in CIT, which responded with shape change and aggregation (mean, 66%). Anticoagulant did not have an effect on convulxin-induced platelet aggregation (mean, ACD-A 65% vs. CIT 69%); however, the slope was significantly less in ACD-A than in CIT.
In washed platelet suspensions at pH 7.4, there were no differences in amplitude of convulxin- or γ-thrombin-induced aggregation at various calcium concentrations, although the slope was greater with increasing [Ca2+] in response to γ-thrombin. Varying pH had no effect on amplitude of aggregation induced by convulxin or γ-thrombin; however, using a lower concentration of γ-thrombin, the amplitude was significantly less at pH 7.0 (mean 40%) than at pH 7.4 (mean 64%). The slope of aggregation increased with rising pH with both agonists.
Canine platelet aggregation induced by ADP and γ-thrombin was negligible in ACD-A PRP, suggesting that increased extraplatelet [H+] inhibits signaling triggered by these agonists but not by convulxin. The choice of anticoagulant may influence in vitro platelet function studies, leading to erroneous conclusions.