Activating Platelets with Thrombin results in a strong activation with 90% of the platelet factor payload being released within 60 mins. The platelets then continue to live for about 8-10 days and manufacture/release lower levels till their energy stores are depleted
When preparing autologous Platelet Rich Plasma for reinjection using Citrate as an anticoagulant, there is likely to be a low level of activation caused by the physical shear forces during centrifugation. I wish to measure this using P-Selectin in a flow cytometer
In principle it doesn't necessarily matter if there is some low level activation provided it hasn't resulted in a great release of the platelet factors before the platelets have been reinjected
However the question I am trying to answer is whether in this passive, accidental activation there is a direct correlation between the activation state as measured by P-Selection and the actual release of Platelet factors during the first 30-60 mins after centrifugation.
Does anybody have any experimental evidence on this?
P selectin's role in platelet is to bind to leukocytes and induce "rolling" in these cells. P-Selectin triggers fibrin formation. As far as I know it does not affect platelet activation, since the platelet has to have been activated to express these molecules.
Citrate is a trigger of intrinsic coagulation. Kallikrein can directly activate prothrombin to thrombin, a strong activator of platelets. To prevent contact phase activation for platelet products to be infused into patients it might be recommendabel to add some arginine to the platelets (that is at about 50-100 mM an efficient inhibitor of intrinsic coagulation).
P-selectin (or GMP-140, PADGEM, CD62P), a member of the selectin family, is a 140-kDaglycoprotein that is a component of the-granule membrane of resting plate-
lets and the Weibel-Palade bodies of endothelial cells.
Into a developing thrombus, It is identified monocyte-derived microparticles in human platelet–poor plasma that express tissue factor.
Tthe first time that platelet-derived membrane vesicles are sufficient to deliver CD154 to stimulate antigen-specific IgG production and modulate germinal center formation through cooperation with responses elicited by CD4+ T cells. The data are consistent with the hypothesis.
Platelet-bound CD154 induces an inflammatory reaction of endothelial cells via CD40.human platelets express CD154 on the cell surface within a very short period of time after activation by thrombin. t platelet-bound CD154 induces an inflammatory reaction of endothelial cells via CD40. Not only thrombin, but also other platelet agonists, can trigger platelets to express CD154 on the cell surface. CD154 expression in intravital thrombi and indicate a link between the formation of a thrombotic plug and the immediate generation of a local inflammatory signal in the affected vessel in vivo.
CD40, the receptor for the CD154 molecule, on the surface of resting platelets. , platelets are capable of co expressing CD154 and its receptor CD40. CD40 has been identified on several cell types in the immune and vascular systems, and its ligation by CD154 usually induces a marked activation of these cells. CD40 on platelets Apparently, binding of CD40 by its ligand is not essential for the haemostatic function of platelets, and this conclusion is indirectly
The interaction of CD154 on stimulated platelets with the coexpressed CD40 receptor leads within minutes to hours to a proteolytic cleavage of CD154 and the release of sCD154, the soluble form of this molecule. The generated 18-kd sCD154 molecule is similar to sCD154 released from activated T cells and can also bind CD40 . ,high levels of sCD154 are not capable of inducing any inflammatory reaction of endothelial cells. Natural sCD154, although capable of binding to CD40-Ig reagents, is relatively unstable.
The measurement of sCD154 allows easy monitoring of platelet activation, and this could be exploited in transfusion-medicine research. Once platelets are strongly activated in the vascular system, CD154 is rapidly expressed on the cell surface of aggregating platelets. So platelet CD154 interacts with CD40 on the neighboring endothelial cells and at the same time with CD40 on monocytes trapped in the thrombus . In both endothelial cells and monocytes, this interaction probably immediately triggers an inflammatory response. These CD154 molecules, which are not engaged by CD40 on other cell types, bind to CD40 co expressed in the platelet aggregate. All of these interactions result over time in the transformation of the proinflammatory form of transmembrane CD154 into the biologically inactive, soluble form of the molecule. The generation of sCD154 takes minutes to hours in vitro, and a similar time scale can be assumed in vivo. , CD154 is located intracellular most of the time and, when translocated to the cell surface following a HLA-complex ,restricted stimulus, only briefly interacts with CD40 on partner cells (eg, B cells). On ligation to CD40, T-cell CD154 is either internalized or shed in a soluble form, with a major sCD154 product resulting from the cleavage between E112 and M113 (single-letter amino acid code).Despite the short duration of CD154 interaction with CD40 on cells of the immune system, the biologic effects of this interaction are a critic role in the switch from IgM to IgG production by B cells . In fact, a very tight regulation of CD154 in the immune system is required because prolonged exposure to a biologically effective form of CD154 leads to a massive inflammation or even triggers the development of lymphomas .Besides with T-cell CD154 are apparently also valid for platelet CD154. Also, it can be assumed that the limited duration of CD154 expression on platelets prevents an excessive inflammation of the endothelium and of CD40+ cells in the vicinity of a hemostatic plug.
A comparison between its structural and functional homologue of TNF-α, platelet CD154 are differs One major difference is the rapidity of action. CD154 molecules exert their inflammatory action in the instant of the formation of a thrombus, whereas the de novo synthesis of TNF-α and other proinflammatory mediators requires several hours. Another important difference is the range of action. The proinflammatory effects of transmembrane CD154 are restricted to the area of platelet aggregation, whereas TNF-α is active in both its transmembrane and soluble forms. The inflammatory signal exerted by platelet CD154 is thus of limited duration. Only when the neighboring endothelial cells and possibly also monocytes start to secrete an array of inflammatory mediators in response to CD40 ligation by platelet CD154 can the local inflammation be regulated and sustained for a prolonged period of time.
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