Regulation of cytosolic calcium by collagen in single human platelets

Abstract

There is controversy in the literature as to whether collagen is able to induce directly a rise in cytosolic calcium concentration ([Ca2+]i) in human platelets. We have addressed this question by observing the cytosolic calcium response of single fura‐2‐loaded human platelets settling onto a collagen‐coated surface using dynamic fluorescence ratio imaging. Following a short lag phase after adherence to collagen fibres, platelets underwent a rapid rise in cytosolic calcium from basal values of 80 ± 13 nM (n = 24) to a peak of 475 ± 42 nM (n = 24) which was sustained for the remaining period of the experiment. The tyrphostin protein tyrosine kinase inhibitor, ST271, reduced substantially the proportion of platelets which exhibited a rise in [Ca2+]i on adherence to collagen and transformed the response in remaining cells to one of oscillations. In contrast, and as a control for collagen, laminin‐coated surfaces induced adherence of human platelets without elevating intracellular [Ca2+]; the cells however remained responsive to ADP. We conclude that collagen directly induces a rise in cytosolic calcium in single human platelets through a tyrosine kinase‐mediated pathway.

Phosphorylation and Activation of Cytosolic Phospholipase A2 by 38‐kDa Mitogen‐Activated Protein Kinase in Collagen‐Stimulated Human Platelets

“…Fcy-IIA cross-linking caused only minimal release of ['Hlarachidonic acid (1 -2 % of total label), which is consistent with the small and transient nature of the evoked intracellular Ca2+ rise [46]. The inhibitors tended to decrease the ['Hlarachidonic acid release, but effects were statistically not significant and could not be compared with those observed in response to collagen.…”

Mechanism of Collagen Activation in Human Platelets

“…Collagen fibers are exposed to circulating platelets following injury to the vessel wall and play an important role in hemostasis through the creation of a physical barrier at the site of vascular damage, thereby limiting blood loss (2)(3)(4), and the stimulation of platelet activation, thereby recruiting additional platelets to the site of damage as well as consolidating the thrombus (4). The dual actions of collagen have been explained in the two-step model for activation, which suggests that adhesion and activation of platelets are mediated through the interaction of collagen with platelet integrin ␣ 2 ␤ 1 and glycoprotein VI, respectively (3, 5).…”

“…Following vascular damage, collagen is exposed to circulating platelets and both acts as a substrate for the adhesion of platelets (2-4) and induces platelet activation (4). The prevailing evidence proposes that two receptors are involved in the platelet response to collagen; integrin ␣ 2 ␤ 1 acts to adhere platelets to collagen, allowing platelets to interact with the lower affinity receptor glycoprotein VI, which is mainly responsible for platelet activation (3,5).…”

Rapid resensitization of purinergic receptor function in human platelets

“…Measurement of cytosolic calcium was performed as previously described [18]. Briefly, 3 lM Fura-2-AM was added to PRP, and incubated at 37°C for 45 min in the presence of 10 lM indomethacin.…”