Dear Gabriel, 

These papers give you some hints:

Article Standardization and Clinical Utility of Thrombin-Generation Assays

Article Assessment of Vascular Endothelial Growth Factor in Fresh ve...

The calcium is required to activate coagulation, resulting in thrombin activation.  Once thrombin is active, calcium itself is not obligatory for thrombin activity.  But, from a simply pragmatic standpoint, there should not be any harm in including the calcium, and it might actually be beneficial (in the sense of avoiding surprises).  Remember that PPP is an extremely complex mixture, and maintaining ionic levels similar to in-vivo concentrations might help avoid unknown or unexpected problems.  Most important with blood studies is to standardize:  never mix conditions that you can control (like having calcium in some experiments but not in others).

That pragmatism is exactly what's making me question this, Craig! Béatrice's suggestions made me change my keywords, and actually found material mentioning that fibrin has calcium binding domains, and they play a role in fibrin polymerization. So I'm with you, being calcium concentration a variable I can manipulate, better safe than sorry and try to maintain in vivo levels.

The calcium Ions bound to fibrin cannot be extracted by citrate, only EDTA or EGTA is strong enough to extract These protein-bound calcium ions.

Thrombin is a potent enzyme and it does not need calcium for triggering the conversion of fibrinogen to fibrin.  In my opinion, there is no need for calcium.

Calcium may not be necessary for thrombin activation per se (see the thrombin time, a coagulation time that is run in non recalcified plasma) or for thrombin mediated platelet activation (mediated by protease activated receptors PARs) or for platelet aggregation (Born aggregation is performed in decalcified plasma).

However, and this can easily demonstrated by flow cytometry, the structure of platelet surface receptors and subsequently the function of these receptors is modified by the presence or absence of calcium. This effect is beautifully demonstrated by Pseudothrombopenia in EDTA. The strong chelator removes all calcium, changing 3D receptor structure, permitting preformed antibodies inthe oatients blood to agglutinate platelets invitro (but not in vivo). So d

epending what you are looking fod, you may indeed want to co trol for calcium concentration.

Intercellular calcium communication regulates

platelet aggregation and thrombus growth

Warwick S. Nesbitt, Simon Giuliano, Suhasini Kulkarni, Sacha M. Dopheide, Ian S. Harper,

and Shaun P. Jackson

Australian Centre for Blood Diseases, Department of Medicine, Monash University, Box Hill Hospital, Victoria 3128, Australia

he ability of platelets to form stable adhesion contacts

with other activated platelets (platelet cohesion or

aggregation) at sites of vascular injury is essential for

hemostasis and thrombosis. In this study, we have examined

the mechanisms regulating cytosolic calcium flux during

the development of platelet–platelet adhesion contacts under

the influence of flow. An examination of platelet calcium

flux during platelet aggregate formation in vitro demonstrated

a key role for intercellular calcium communication

T (ICC) in regulating the recruitment of translocating platelets

into developing aggregates. We demonstrate that ICC is

primarily mediated by a signaling mechanism operating

between integrin IIb3 and the recently cloned ADP purinergic

receptor P2Y12. Furthermore, we demonstrate that the

efficiency by which calcium signals are propagated within

platelet aggregates plays an important role in dictating the

rate and extent of thrombus growth.

 Curr Mol Med. 2012 Dec;12(10):1319-28.

Transient receptor potential channels in human platelets: expression and functional role.

Dionisio N1, Redondo PC, Jardin I, Rosado JA.

Author information

Abstract

Recent studies have demonstrated that mammalian homologues of Drosophila transient receptor potential (TRP) channels are widely expressed in human platelets. Occupation of G protein-coupled receptors by agonists results in activation of these channels, which results in Na+ and Ca2+ entry. Canonical or classic TRP (TRPC) family members have been reported to associate with different Ca2+-handling proteins, including the type II inositol 1,4,5-trisphosphate receptor, the endoplasmic reticulum Ca2+ sensor STIM1 (STromal Interaction Molecule-1) or the Ca2+ permeable channel Orai1. The dynamic interaction of TRPC channels with the above mentioned proteins has been found to be important for both store-operated and capacitative Ca2+ entry, as well as for non-capacitative Ca2+ influx. The former is a major mechanism for Ca2+ entry in human platelets. This mechanism, activated by a reduction in the concentration of free Ca2+ in the intracellular stores, results in the formation of signaling complexes involving STIM proteins, Orai1, Orai2, TRPC1 and TRPC6. There is a growing body of evidence supporting that Ca2+ signaling dysfunction plays an important role in the pathogenesis of several platelet-linked disorders, including those associated to type 2 diabetes mellitus. Abnormal Ca2+ signals in response to physiological agonists have been associated to platelet hyperactivity. The expression of several TRPCs, STIM1 and Orai1, as well as their interaction, has been reported to be altered in platelets from type 2 diabetic patients, which results in attenuated capacitative Ca2+ entry but enhanced non-capacitative Ca2+ influx; thus suggesting a role for Ca2+ handling proteins, including TRPs, in the pathomechanism of diabetic complications

 As the two articles I just sent show, calcium has a role in platelet aggregation iv vivo. Therefore, while thrombin without calcium will cause platelets to aggregate, adding calcium within will be more physiological.

EDTA is very potent chelator and may block the activation of thrombin.

From pragmatic standpoint I suggest doing double test with- and without additive calcium.

EDTA blocks thrombin activation but does not block thrombin activity once activated. he is using activated IIa.

Calcium is needed in coagulation cascade as it is a cofactor for membrane-bound complexes, including intrinsic tenase (FIXa-FVIIIa),extrinsic tenase (FVIIa-TF) and prothrombinase (FXa-FVa) complex. The prothrombinase complex cleaves prothrombin to thrombin. Once thrombin is generated, it can cleave fibrinogen to fibrin or even activate platelet in a calcium-independent manner.

However, some questions arise:

1. How do you know that you have thrombin? How do you measure?

2. What is the anticoagulant you use when you collect whole blood?