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

BACKGROUND: 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. OBJECTIVES: To define anoctamin proteins and scramblase response of CSS platelets and to determine whether TMEM16F is the CSS disease gene. METHODS: CSS TMEM16F cDNA and gene were sequenced and mutation detection was performed in CSS pedigrees. Platelet fractions from CSS dogs were isolated for proteomic and immunologic characterization of TMEM16F. Annexin V was used as a flow cytometric marker of induced platelet PS externalization. RESULTS: A TMEM16F splice site mutation segregated with the CSS trait and TMEM16F protein was undetectable in CSS platelet membranes; however, a second anoctamin, TMEM16K, was found. Proteomic analyses revealed a network of 32 proteins that differentially cosegregated with platelet plasma membrane TMEM16F. CSS platelets had profoundly impaired scramblase response to pharmacologic and physiologic agents that increase intraplatelet calcium and conditions that induce apoptotic and necrotic cell death. CONCLUSIONS: CSS platelets represent a TMEM16F-null mutant model that demonstrates a central role for TMEM16F in mediating platelet PS externalization in response to activating and death signals. Platelet TMEM16F may prove a novel drug target for modulating platelet procoagulant activity and extending platelet life span.

Statin drugs and dietary isoprenoids downregulate protein prenylation in signal transduction and are antithrombotic and prothrombolytic agents.

Statins and various isoprenoids of dietary origins inhibit L-mevalonic acid synthesis, which in turn downregulates cholesterol and various other dependent substances, including farnesyl- and geranylgeranyl-conjugated proteins involved in cell signaling processes. Such signaling processes are stimulated by protease-activated receptor-1 (PAR-1), which upon activation, causes the expression of various substances including tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1). Tissue factor promotes thrombin generation, where thrombin stimulates a variety of cellular processes, as well as activating PAR-1 to produce more thrombin. Statins downregulate TF mitigating thrombin generation and also downregulate PAI-1, which normally consumes tissue plasminogen activator (tPA). In the absence of PAI-1, tPA activates plasminogen to generate plasmin. Thus, statins behave as antithrombotic agents and prothrombolytic agents.

Molecular and genetic basis for thrombasthenic thrombopathia in otterhounds.

OBJECTIVES: To determine the molecular and genetic basis for thrombasthenic thrombopathia in Otterhounds and establish whether the defect would be best classified as type-I Glanzmann's thrombasthenia. ANIMALS: 57 dogs, including 13 affected Otterhounds, 23 carrier Otterhounds, 17 unaffected Otterhounds, and 4 clinically normal unrelated dogs of other breeds. PROCEDURE: Functional (platelet aggregation, clot retraction, buccal mucosa bleeding time) and biochemical (electrophoresis, flow cytometry, fibrinogen content) analyses were conducted. In addition, first-strand cDNA synthesis from platelet total RNA was performed. Exons of the genes encoding for glycoproteins (GP) IIb and IIIa were amplified in overlapping fashion. The resulting products were excised from agarose gels and sequenced. The sequences obtained were compared with known cDNA sequences for canine GPIIb and GPIIIa. RESULTS: A single nucleotide change at position G1193 (1100) was detected in exon 12 of the gene encoding for platelet GPIIb in 2 affected Otterhounds. Carrier Otterhounds were heterozygous at this position, and 2 unaffected Otterhounds were unchanged. This nucleotide change would result in substitution of histidine for aspartic acid at position 398 (367) within the third calcium-binding domain of GPIIb. CONCLUSIONS AND CLINICAL RELEVANCE: These studies suggest that thrombasthenic thrombopathia of Otterhounds is homologous phenotypically and has a similar molecular basis to type-I Glanzmann's thrombasthenia in humans.

Platelet thrombus formation in microvessels of young spontaneously hypertensive rats.

We have previously shown an increase in platelet-to-endothelial cell adhesion in microvessels of spontaneously hypertensive rats (SHR) during the established stage of hypertension (12 weeks). The objective of the current study was to determine if the platelet-to-endothelial cell interaction would be altered in the early developmental phase of hypertension. Male weanling (3 weeks old) SHRs (n=6) and age matched normotensive Wistar-Kyoto (WKY) rats (n=6) were used to study platelet thrombus formation. Intravascular fluorescein isothiocyanate tagged to bovine serum albumin was activated with 450-490 nm light to induce thrombus formation in microvessels. Plasma concentrations of von Willebrand factor (vWF), fibrinogen and fibronectin (FN) were measured in rats during both early (3 week) and established stages of hypertension development. Thrombus initiation time in both arterioles (847+/-85 sec) and venules (222+/-40 sec) of young SHRs was significantly shorter (p<0.05) than in arterioles (1270+/-88 sec) and venules (630+/-72 sec) of age matched WKY rats respectively. After thrombus appearance, however, overall time for vessel occlusion in arterioles (2590+/-90 sec) and venules (935+/-131 sec) of SHRs was not different compared to that in arterioles (2650+/-191 sec) and venules (1240+/-93 sec) of age matched WKY rats. The plasma concentration of FN was increased (p<0.05) in both the young (0.9+/-0.1 mg/ml) and mature (1.1+/-0.2 mg/ml) hypertensive rats (n=5) compared to that in young (0.6+/-0.03 mg/ml) and mature (0.5+/-0.1 mg/ml) WKY rats (n=5), while fibrinogen content (3.6 +/-0.3 mg/ml) was elevated (p<0.05) only in mature SHRs (n=5) compared to that (2.7+/-0.02 mg/ml) in age matched WKY rats (n=5). The plasma concentration of vWF was similar to that of controls in either age group of hypertensive animals. These results suggest that changes in platelet-to-endothelial cell interactions occur in the early phase of genetic hypertension development in rats, and appears to result from alteration of plasma concentration of adhesion proteins.

Factor X deficiency in a cat.

Severe congenital deficiency of factor X was diagnosed in a 3-year-old castrated male domestic shorthair cat with clinical signs of generalized seizures and prolonged bleeding after venipuncture. Heritability of factor X deficiency was suspected because of a prolonged Russell's viper venom time in the dam and reductions in factor X activity in the dam and 1 sibling. To our knowledge, factor X deficiency in cats has not been reported previously. Definitive diagnosis for animals with clinical signs of coagulopathy may require repetition of coagulation screening tests using different assay methods or specific coagulation factor analyses.

Von Willebrand factor restores impaired platelet thrombogenesis in copper-deficient rats.

Dietary copper restriction reduces microvascular thrombogenesis. We have now examined the roles of shear forces and von Willebrand factor (vWF) in in vivo thrombus formation in the cremaster microcirculation of copper-deficient rats. Male weanling Sprague-Dawley rats were fed purified diets that were either copper-adequate (6.3 mg Cu/kg) or copper-deficient (0.3 mg Cu/kg) for 4 wk. Intravascular fluorescein isothiocyanate tagged to bovine serum albumin was activated with 450-490 nm light to induce thrombus formation in microvessels. Thrombus initiation time was significantly prolonged in copper-deficient rats; after thrombus appearance, however, vessel occlusion was significantly accelerated. The greater shear rates of arterioles compared with venules significantly increased the thrombus initiation time in both groups. However, vessel occlusion time and thrombus growth time were independent of shear rate. Intravascular vWF (0.2 u/100 g body wt) decreased thrombus initiation time in the CuD group without affecting thrombus growth time. The data suggest that decreased thrombogenesis in copper-deficient rats is not a result of altered rheological factors or arteriolar-venular differences, but appears to result from decreased platelet-to-endothelial cell adhesion.

Platelet aggregation and adhesion during dietary copper deficiency in rats.

The role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 micrograms copper/g of diet) or copper-deficient (CuD, 0.3 microgram/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rate endothelial cells. After a one hour incubation at 37 degrees C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 x 10(-4)M) and measured in a turbidometric aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from ++CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.

Bleeding diathesis associated with variant von Willebrand factor in a Simmental calf.

A 10-month-old Simmental heifer was examined because of a 10-day history of epistaxis and aural hematomas. Examination of the calf also revealed hemarthrosis. Initial laboratory data indicated that platelet count, platelet size, prothrombin time, and partial thromboplastin time were not different from a clinically normal (control) cow. Mucosal bleeding time was prolonged, and platelet adhesion to glass beads was less than expected. The clinical signs, prolonged bleeding time, and platelet adhesion defect were corrected by infusion of bovine plasma. Subsequent laboratory testing revealed that the affected calf had a truncated multimeric structure of von Willebrand factor (vWF), low vWF activity, and impaired platelet aggregation in response to adenosine diphosphate, but concentration of vWF was not different from that in clinically normal control animals. These data were consistent with a diagnosis of variant von Willebrand disease. The relationship of this disease to the thrombopathy of Simmental cattle is unclear.

A multispecies enzyme-linked immunosorbent assay for von Willebrand's factor.

A modified double-sandwich enzyme-linked immunosorbent assay (ELISA), which was developed for human and canine von Willebrand's factor (vWF), was adapted for quantitation of vWF in other species. In addition to human and dog plasmas, 12 other mammalian plasmas that were surveyed exhibited significant cross-species reactivity with antibodies specific for canine vWF or monoclonal antibodies against porcine or bovine vWF. Mixed combinations of monoclonal antibodies and various polyclonal antibodies were also used as sandwich or capture antibodies. The ability of this multispecies ELISA to detect less than 0.002 U of vWF per milliliter of plasma in a large number of species enhances its utility for both research and clinical diagnostic applications. A quantitative assay for rabbit vWF, which exhibits poor cross-species reactivity with most antibodies, was constructed with anti-porcine monoclonal antibody W1-5 and goat-anti-canine vWF as capture and sandwich antibodies, respectively. The same conjugate antibody configuration was used to visualize rabbit vWF multimers by immunoblotting. Specificity of the assay for vWF in human, dog, pig, and horse plasmas was confirmed by use of species-specific vWF-deficient plasmas. In other species, for which vWD plasmas were not available, ELISA specificity for vWF was demonstrated by recovery of greater than 75% of the ELISA-reactive antigen coincident with vWF multimers in the high-molecular-weight (greater than 500 kd) fractions of purified plasmas. This multispecies ELISA permits, for the first time, the measurement of vWF in a variety of mammals for which species-specific immunologic reagents do not currently exist. The results also suggest that certain vWF epitopes have been highly conserved among phylogenetically diverse species.

A sensitive immunoassay for von Willebrand factor.

We have developed an ELISA specific for canine von Willebrand factor antigen (vWF:Ag) that also strongly reacts with the VWF:Ag of humans and many other vertebrates. This assay was designed to avoid the use of immunoreagents of human origin, however, commercially available antibodies to human vWF:Ag may also be used. von Willebrand factor (vWF) was quantitated using a modified double-sandwich ELISA with polyclonal antibodies specific for canine vWF:Ag. The assay was as sensitive for measuring canine vWF:Ag as previously published immuno-radiometric assays and the most sensitive ELISA for human vWF:Ag. Employing commercially available antibodies to human vWF:Ag in the same double-sandwich configuration, the lower limit of detection for human vWF:Ag was 4.8 x 10(-6) units/ml, lower by a factor of ten than previously reported ELISAs. In addition, a wide range of vWF:Ag levels can be determined with just a single plasma dilution. The assay readily distinguishes type III von Willebrand disease from other types of von Willebrand disease having very low levels of vWF. This vWF ELISA can be used to evaluate large numbers of plasma samples simultaneously and is therefore well-suited for large-scale screening programs.

Thrombin inhibition with dipeptidyl argininals.

Human alpha- and gamma-thrombins (with high and essentially no fibrinogen clotting activities, respectively) were inhibited in chromogenic substrate assays by the dipeptidyl argininals: antipain less than leupeptin less than H-D-Phe-Pro-argininal approximately Boc-D-Phe-Pro-argininal. In clotting assays with alpha-thrombin, I50 values were slightly higher than Ki values from chromogenic substrate assays, except for a somewhat lower I50 for antipain. Our data cautions the use of argininal proteinase inhibitors in the assessment of thrombin functions, and the high potency of H-D-Phe-Pro-argininal and its derivative suggest pharmaceutical applications.

Complement C5a (desArg) generation in serum exposed to damaged aortic endothelium.

Complement activation by injured endothelial cells was investigated using ex vivo rabbit thoracic aortas as a source of endothelium and a neutrophil aggregation (NA) bioassay to detect the complement cleavage product C5a (desArg). Endothelium, oxygen-starved by incubating aortas 30 min with Tyrode's buffer, activated complement as demonstrated by a 57% increase in the NA response induced by serum from buffer-treated aortas as compared to serum from untreated control aortas. Incubation of MgEGTA serum in injured aortas resulted in a 27% (P less than 0.025) weaker NA response than normal serum, indicating participation by both classical and alternative pathways of complement activation. Serum from aortas incubated 30 min with 100 micrograms/ml cholestane-3 beta, 5 alpha, 6 beta-triol, a cytotoxic cholesterol oxidation derivative, induced a NA response comparable to that from serum from aortas treated 30 min with Tyrode's buffer. Heat inactivation of serum prior to aortic incubation abolished NA activity and serum incubated in deendothelialized aortas lacked NA activity. Fractionation of serum samples from these experiments on Sephadex G-100 revealed a single peak of NA activity corresponding to the molecular weight of C5a (desArg). Endothelial cell injury was demonstrated by the inability to exclude Trypan blue dye and by scanning electron microscopy. These data demonstrate that damaged arterial endothelium can effectively activate the complement system, resulting in the production of an anaphylatoxic inflammatory mediator.

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.

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.

Complement activation after aortic endothelial injury.

Ischemic endothelial cell injury results in C activation that precedes partially via the classic pathway. This may be relevant to clinical situations of ischemia such as occur during embolization or thrombus formation. The generation of C activation products (such as C3a and C5a) may mediate many of the inflammatory changes observed in infarcted tissues.