Dual agonist stimulation of platelets with thrombin plus collagen, or thrombin plus convulxin (a ligand of the collagen receptor glycoprotein VI), generates a subpopulation of highly procoagulant cells known as ‘coated’ platelets . Coated-platelets represent approximately 30% of the total platelet population and display a combination of characteristic features including expression of phosphatidylserine (PS), surface retention of alpha granule proteins, permeability to calcein, and release of membrane-derived microparticles [2–6]. Furthermore, recent studies demonstrate that loss of mitochondrial membrane potential via formation of a mitochondrial permeability transition pore (MPTP), a key feature of the intrinsic apoptotic pathway, is also an integral event leading to platelet membrane PS externalization and coated platelet formation . While in vitro experimental systems indicate that the proportion of coated-platelets correlates with thrombin generation [3,5], the role of coated-platelets in physiologic and pathologic thrombus formation in vivo is still under investigation.
In a series of experiments, we investigated the formation of coated-platelets utilizing a canine model of Scott syndrome. This trait was identified in a colony of German shepherd dogs (GSD) with an autosomal recessive bleeding diathesis characterized by a specific deficiency of platelet procoagulant activity . Affected GSD demonstrate the pathognomonic platelet phenotype of Scott syndrome (i.e. a lack of PS expression and failure of membrane microvesiculation upon activation with calcium ionophore) [8,9]. Furthermore, Scott GSD platelets do not generate prothrombinase activity in response to ionophore or the physiologic agonists thrombin and collagen . We hypothesized that dual agonist stimulation of Scott GSD platelets would not only fail to elicit PS externalization, but that Scott GSD platelets would be incapable of displaying the characteristic features of coated-platelets .
Coated-platelets were produced by thrombin plus convulxin stimulation of platelet-rich plasma (PRP) prepared from healthy control dogs and clinically affected GSD. All activation experiments were performed in a 100 μL (total) assay volume containing 1 μL PRP and the following reagents (final concentrations): 1 mg mL−1 BSA, 2 mm CaCl2, 1 mm MgCl2, 1 μg mL−1 biotin-fibrinogen, 500 ng mL−1 convulxin, 0.5 U mL−1 bovine thrombin, 0.4 mm gly-pro-arg-pro-NH2, 150 mm NaCl, and 10 mm HEPES, pH 7.5. The reaction tubes were incubated at 37° for 10 min, and individually labeled, as previously described [4,6,7,10], with the following specific markers of coated-platelets: Annexin V-fluorescein isothicyanate (FITC) to detect PS expression; phycoerythrin-streptavidin and FITC-abciximab to label surface bound biotinylated fibrinogen and identify platelets; calcein-AM to detect calcein release; and JC-1 to monitor the loss of mitochondrial membrane potential, denoting formation of the MPTP.
Scott GSD platelets failed to bind fibrinogen, externalize PS or release calcein upon dual agonist stimulation, in contrast to the responses observed for control platelets (Fig. 1 and Table 1). However, there were no significant differences between Scott GSD and control dogs in the percentages of stimulated platelets demonstrating a shift in JC-1 fluorescence (Table 1). These data demonstrate that the platelet phenotype of canine Scott syndrome comprises a failure to generate coated-platelets in response to dual agonist stimulation, in spite of an apparently normal formation of MPTP. Previously we have defined coated-platelets by the retention of surface-bound fibrinogen  with the recognition that other intermediate markers of coated-platelet formation (i.e. MPTP, calcein release and PS exposure) are required events but not sufficient to produce coated-platelets .
|Subjects||n||Flow cytometry markers|
|% Positive cells||% Negative cells|
|Control dogs||5||37.3 ± 6.1||53.9 ± 10.4||36.6 ± 5.6||60.6 ± 10.1|
|Scott GSD||5||5.1 ± 2.2**||4.2 ± 3.9**||3.3 ± 0.9**||65.3 ± 8.6|
Our previous studies of Scott GSD revealed normal aggregation, secretion, and clot retraction in response to thrombin and/or collagen stimulation , indicating that the receptors and signaling pathways for these agonists remain intact and that the glycoprotein IIb/IIIa complex on these platelets is functional. In the current study, we observed a complex pattern of abnormalities in markers of coated-platelets (Table 1). As pedigree studies of the Scott GSD colony indicate a simple autosomal recessive trait , it is likely that all these anomalies are caused by a single gene defect. One possible explanation for this finding is that a pathway of sequential events is required for coated-platelet formation and that interruption of any upstream event abolishes all downstream events. In this model, the combined stimulation of thrombin and collagen receptors induces a loss of mitochondrial membrane potential in only a subset of platelets. The formation of MPTP in this subset of cells results in the release of cytoplasmic calcein, externalization of PS and production of coated-platelets as indicated by fibrinogen retention. According to this model, the Scott GSD platelet defect is downstream of MPTP formation, but upstream of calcein release, PS exposure and fibrinogen retention. In support of this proposal, pharmacologic inhibition of MPTP formation  has been shown to prevent calcein loss, PS externalization and coated-platelet formation. Similarly, genetic disruption of MPTP formation  results in a failure of PS exposure and coated-platelet formation; calcein-release in this system has yet to be measured.
Certain phenotypic features of Scott GSD differ from human Scott syndrome patients. In contrast to human Scott cells [9,12], Scott GSD have no demonstrable abnormality in red cell PS externalization  or in platelet expression of the lipid transporter, ABCA-1 . The hallmark of Scott syndrome, an inability to express PS on activated platelets, may therefore result from more than a single molecular defect. Nevertheless, Scott GSD platelets provide a unique model system to further elucidate the pathways leading to stimulated PS externalization and the role of platelet membrane PS in localization and amplification of thrombin generation.