James L. Catalfamo

Scott syndrome dogs have impaired coated-platelet formation and calcein-release but normal mitochondrial depolarization

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 [1]. Coatedplatelets 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 [6]. 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 [7]. 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 [7]. 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 [1]. Coated-platelets were produced by thrombin plus convulxin stimulation of platelet-rich plasma (PRP) prepared from healthy control dogs and clinically affectedGSD.All activation experiments were performed in a 100 lL (total) assay volume containing 1 lL PRP and the following reagents (final concentrations): 1 mg mL BSA, 2 mM CaCl2, 1 mM MgCl2, 1 lg mL )1 biotin-fibrinogen, 500 ng mL convulxin, 0.5 U mL bovine thrombin, 0.4 mM gly-pro-arg-pro-NH2, 150 mMNaCl, and 10 mMHEPES, 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

[4] Isolation of platelets from laboratory animals

Publisher Summary Known procedures for the separation of human platelets from peripheral blood are frequently adapted with variable success for the isolation of platelets from laboratory animal species. This chapter describes the process of isolating platelets from laboratory animals. Successful platelet isolation depends on the quality of the blood sample, which is influenced by the collection technique, the general health of the animal, and the uniformity of the subgroup or strain used. The techniques required to isolate platelets from the peripheral blood of all seven species are similar. Only blood samples obtained using a two-syringe technique and with minimal tissue trauma are suitable for platelet preparation. Platelet-rich plasma from all seven species can be used to obtain gel-filtered platelets (GFPs). The platelets are separated on a 1.5 × 30-cm column packed with acetone-washed Sepharose 4B containing 0.35% of bovine albumin. Following filtration, the GFP preparation is made 1 mM in CaCl. Under these conditions, more than 85% of platelet recovery can be expected from platelet-rich plasma (PRP). Human or species-specific fibrinogen is added to the GFP in most aggregation studies.

Impaired cAMP metabolism associated with abnormal function of thrombopathic canine platelets

The effects of forskolin and 1-Methyl-3-isobutyl-xanthine on thrombopathic platelet function and cyclic AMP accumulation were examined. The concentration of forskolin required to inhibit gamma-thrombin- induced platelet aggregation and secretion by 50% was significantly lower for thrombopathic than for normal platelets. This inhibition was accompanied by a marked elevation of cyclic AMP. 1-Methyl-3-isobutyl-xanthine, alone or in combination with forskolin, augmented both the cyclic AMP accumulation and the inhibition of platelet function. These results demonstrate that cyclic AMP metabolism is abnormal in thrombopathic platelets and imply that cyclic AMP-phosphodiesterase activity is impaired.

A Genome-wide Linkage Scan in German Shepherd Dogs Localizes Canine Platelet Procoagulant Deficiency (Scott Syndrome) to Canine Chromosome 27

Scott syndrome is a rare hereditary bleeding disorder associated with an inability of stimulated platelets to externalize the negatively charged phospholipid, phosphatidylserine (PS). Canine Scott syndrome (CSS) is the only naturally occurring animal model of this defect and therefore represents a unique tool to discover a disease gene capable of producing this platelet phenotype. We undertook platelet function studies and linkage analyses in a pedigree of CSS-affected German shepherd dogs. Based on residual serum prothrombin and flow cytometric assays, CSS segregates as an autosomal recessive trait. An initial genome scan, performed by genotyping 48 dogs for 280 microsatellite markers, suggested linkage with markers on chromosome 27. Genotypes ultimately obtained for a total of 56 dogs at 11 markers on chromosome 27 revealed significant LOD scores for 2 markers near the centromere, with multipoint linkage indicating a CSS trait locus spanning approximately 14 cm. These results provide the basis for fine mapping studies to narrow the disease interval and target the evaluation of putative disease genes.

Flow cytometric detection and procoagulant activity of circulating canine platelet-derived microparticles.

OBJECTIVE To measure platelet membrane-derived microparticle (PMP) content and thrombin-generating capacity of canine plasma subjected to specific processing and storage conditions. ANIMALS 31 clinically normal dogs (19 males and 12 females). PROCEDURES Citrate-anticoagulated blood samples obtained from each dog were centrifuged at 2,500 × g to isolate platelet-poor plasma (PPP), then PPP was centrifuged at 21,000 × g to isolate microparticle-free plasma (MPF) and microparticle-enriched plasma (MPEP). Whole blood and paired samples of fresh and frozen-thawed PPP, MPF, and MPEP were dual labeled for flow cytometric detection of membrane CD61 (constitutive platelet antigen) and annexin V (indicating phosphatidylserine externalization). Platelets and PMPs were enumerated with fluorescent, size-calibrated beads. Thrombin generation in fresh and frozen-thawed PPP, MPF, and MPEP was measured via kinetic fluorometric assays configured with low tissue factor and low phospholipid concentrations. RESULTS Initial centrifugation yielded PPP with < 0.5% the platelets of whole blood, with median counts of 413 PMPs/μL for males and 711 PMPs/μL for females. Sequential centrifugation resulted in a 10-fold concentration of PMPs in MPEP and virtually depleted PMPs from MPF. Thrombin generation depended on PMP content, with median endogenous thrombin potential of 0, 893, and 3,650 nmol•min for MPF, PPP, and MPEP, respectively. Freeze-thaw cycling caused significant increases in PMP counts and phosphatidylserine externalization. CONCLUSIONS AND CLINICAL RELEVANCE Canine PMPs were major determinants of thrombin-generating capacity; preanalytic variables influenced plasma PMP content. Processing conditions described here may provide a basis for characterization of PMPs in clinical studies of thrombosis in dogs.

A TMEM16F point mutation causes an absence of canine platelet TMEM16F and ineffective activation and death-induced phospholipid scrambling.

TMEM16F is an ion channel and calcium‐dependent lipid scramblase that mediates phosphatidylserine (PS) exposure on the plasma membrane. Two disparate disease phenotypes are associated with TMEM16F loss‐of‐function mutations: a rare bleeding disorder (Scott syndrome) and skeletal malformations due to aberrant bone mineralization in a TMEM16F knockout mouse. We therefore undertook comparative studies of TMEM16F expression in canine Scott syndrome (CSS), an autosomal recessive platelet defect.

Intravenous Shiga toxin 2 promotes enteritis and renal injury characterized by polymorphonuclear leukocyte infiltration and thrombosis in Dutch Belted rabbits.

Enterohemorrhagic Escherichia coli (EHEC) infection causes hemolytic uremic syndrome, a leading cause of acute renal failure in children. Dutch Belted (DB) rabbits are susceptible to EHEC-induced disease. Using real-time quantitative RT-PCR we measured the renal mRNA expression of cytokines and fibrinolytic factors in DB rabbits challenged with intravenous Shiga toxin 2 (Stx2) (1200 ng/kg). Group 1 rabbits received an incremental dose during an 8-day period whereas Group 2 rabbits received a single dose. Group 1 rabbits developed mild disease. In contrast, Group 2 rabbits developed severe diarrhea, higher levels of circulating polymorphonuclear leukocytes, increased mean platelet volume, and increased fibrinogen levels. Group 2 rabbits developed polymorphonuclear leukocyte infiltration in the intestine and kidney as well as glomerular congestion, luminal constriction, and mesangial glomerulonephropathy. These renal lesions were associated with up-regulation of interleukin-8 (P<0.006), plasminogen activator inhibitor-1 (P<0.04), and tissue plasminogen activator (P<0.05). Circulating Stx2 promoted dose-dependent enteritis and renal injury characterized by inflammation and impaired fibrinolysis leading to thrombosis.

Hereditary and Acquired Thrombopathias

Normal platelet function is essential for hemostasis. The pathophysiologic processes that result in acquired quantitative platelet abnormalities are detailed. Platelet pathophysiology associated with acquired qualitative disorders, either drug-induced or secondary to disease, is discussed. Numerous hereditary platelet defects also exist. Their functional and biochemical features are presented.

Current Diagnostic Trends in Coagulation Disorders Among Dogs and Cats

The diagnostic workup to differentiate hemorrhage caused by vascular injury from a systemic hemostatic imbalance typically involves a combination of broad screening tests and specific assays. The characterization of 3 overlapping phases of primary hemostasis, secondary hemostasis, and fibrinolysis provides a simple diagnostic framework for evaluating patients with clinical signs of hemorrhage. New techniques such as flow cytometry, thrombin-generation assays, thrombelastography, and anticoagulant drug monitoring are under investigation for veterinary patients; however, their ability to improve diagnosis or treatment requires further study in clinical trials.

Equid Herpesvirus Type 1 Activates Platelets

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.