A vital function of platelets is to adhere to a site of vascular injury where collagen and other extracellular matrix proteins, including tissue factor, are exposed to flowing blood. The platelets provide a cellular surface for localized thrombin generation. In the past, platelets were considered a homogenous population with equal potential for activation, which would lead to stimulation of local generation of thrombin. However, recent evidence suggests that all activated platelets are not equal and that a subset (ie, coated platelets) that can be observed after combined stimulation with dual agonists (ie, collagen and thrombin) have a unique potential for retaining procoagulant activity on their surface.1
Coated platelets are defined as platelets with high concentrations of α-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, and coagulation factor V,2 retained with exceptional affinity on their surfaces1,3 via serotonin-mediated coupling by means of transglutaminase activity.4,5 Physiologic agonists of platelets (eg, ADP, which is widely available in platelets and erythrocytes) are able to activate platelets as assessed by use of traditional markers of platelet activation (eg, P-selectin) and to increase the amount of fibrinogen binding, although this binding is reversible and is only at a low degree. Uniquely, formation of a subpopulation of platelets with the coated phenotype (eg, characterized by high fibrinogen binding) requires the combined stimulation of thrombin and collagen or convulxin. Even when maximal externalization of phosphatidylserine on all platelets was induced via nonphysiologic stimulation with an ionophore, only a subpopulation of platelets had the coated phenotype with high externalization of α-granule protein.2 The enhanced procoagulant potential of coated platelets is used in the pharmacological application of recombinant human coagulation factor VIIa, which binds preferentially to coated platelets.6
The complexity of platelet activation and platelet-protein interactions complicates research on platelet physiologic processes and platelet-related disease. This poses a problem with regard to diagnosis in veterinary medicine and also in translational research that is intended to use knowledge from naturally occurring diseases in dog populations as a means for studying diseases in humans. Several relevant diseases (eg, hemophilia A and B,7,8 coagulation factor VII deficiency9–11 von Willebrand disease,12 Glanzmann thrombasthenia,13,14 platelet dense [δ] granule defect,15 and Scott syndrome16,17) exist in dogs. Furthermore, undiagnosed platelet defects in dogs, some of which may be relevant to diseases in humans, may be identified through the use of more advanced biomolecular tools.18
To correctly characterize platelet disorders and accurately assess the procoagulant potential of canine platelets, a broader panel of surface markers for platelet activation is needed. Such a panel would potentially allow detection of extremely subtle differences in platelet activation. Traditionally, expression of P-selectin on the surface of platelets has been used as a measure of platelet activation. Stimulation of canine platelets with a single agonist has been used for assessment of maximal platelet expression of P-selectin.17,19 For human platelets, no distinct patterns for expression of P-selectin were identified in experiments that revealed differential binding of coagulation factors to coated and non-coated platelets.3 Therefore, more subtle distinctions between P-selectin expression and additional markers of platelet activation may facilitate a better understanding of the complex series of biological events that lead to platelet activation and enhanced procoagulant activity of platelets.
We hypothesized that formation of coated platelets in dogs could be identified via externalization of platelet fibrinogen by use of an antibody-based flow cytometric assay, as has been described in humans.20,21 Furthermore, we hypothesized that recombinant human coagulation factor VIIa would bind preferentially to the population of coated platelets in dogs. The purpose of the study reported here was to assess by use of a flow cytometric assay the fibrinogen externalization from platelet α-granules and compare it with P-selectin expression for conditions that involved the use of platelet-activating stimuli similar to those expected to be relevant at the site of a vascular injury. We further modified the assay so that we could assess the mechanism of action for recombinant human coagulation factor VIIa and determine whether it is comparable in humans and dogs.
Active site-inhibited recombinant human coagulation factor VIIa
ABX Micros ABC Vet, HORIBA ABX, Irvine, Calif.
Sigma-Aldrich, St Louis, Mo.
PE-conjugated monoclonal mouse anti-human CD61, clone Y2/51, Santa Cruz Biotech, Santa Cruz, Calif.
Monoclonal mouse anti-human CD62P, clone 1E3, Santa Cruz Biotech, Santa Cruz, Calif.
Polyclonal rabbit anti-human fibrinogen, A0080, Dako, Glostrup, Denmark.
Lightning-Link PerCP Conjugation Kit, Innova Biosciences, Cambridge, England.
Lightning-Link APC Conjugation Kit, Innova Biosciences, Cambridge, England.
Normal mouse IgG2a K, BD, Franklin Lakes, NJ.
Rabbit normal IgG, X0903, Dako, Glostrup, Denmark.
PE-conjugated normal mouse IgG1, Santa Cruz Biotech, Santa Cruz, Calif.
Annexin V-FITC, 556419, BD Pharmingen, Franklin Lakes, NJ.
Pentapharm, Basel, Switzerland.
Enzyme Research, Swansea, Wales.
Instrumentation Laboratory, Lexington, Mass.
Dako CyAn ADP cytometer configured with Dako Summit, version 4.3, Dako, Glostrup, Denmark.
FCS Express 3.0, De Novo Software, Los Angeles, Calif.
Rainbow calibration particles, 8 peaks, 3.0 to 3.4 μm, BD, Franklin Lakes, NJ.
GraphPad Prism, version 5.02, GraphPad Software Inc, La Jolla, Calif.
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