Thrombosomes: a platelet-derived hemostatic agent for control of noncompressible hemorrhage.

BACKGROUND: Uncontrolled hemorrhage is responsible for ∼80% of the potentially survivable deaths in combat and over 40% of early mortality in the under 65 age group in the United States. Providing an easily used infusible hemostatic agent to first responders could significantly reduce these fatalities. We report on an infusible lyophilized platelet-derived hemostatic agent stabilized with trehalose and polysucrose prior to and during lyophilization. STUDY DESIGN AND METHODS: Characterization included determining the particle population size range, surface marker expression GPIb, GPIIbIIIa, and Annexin V binding. Function was assessed by aggregation, thromboelastography, and thrombin generation. Pharmacokinetics, biodistribution, and immunogenicity established using Indium(111) labeled Thrombosomes in healthy New Zealand white rabbits (NZWRs), efficacy in thrombocytopenic NZWR, and safety in NZWRs, canines, and nonhuman primates. RESULTS: Thrombosomes retained GPIIbIIIa expression (98.71% ± 0.18 of the rehydrated particles), a reduced expression of GPIb (47.77% ± 6.65), and Annexin V binding (86.05% ± 2.65). Aggregation to all agonists except thrombin in buffer (78.15% ± 2.5) was <50%. Thrombin generation and thromboelastography results demonstrated a concentration gradient that was consistent from lot to lot. There were no observed adverse events in any safety study and blood loss was reduced by >80% in the thrombocytopenic ear bleed model. CONCLUSION: Our in vitro characterization studies in conjunction with preclinical animal safety and efficacy studies demonstrated lot consistency in manufacturing, maintenance of hemostatic functions of Thrombosomes, safety at high dose concentrations, and the potential to provide an effective hemostatic agent at the site of injury.

Two classes of anti-platelet drugs reduce anatomical infarct size in monkey hearts.

BACKGROUND: Recent studies in rabbits have demonstrated that platelet P2Y12 receptor antagonists are cardioprotective, and that the mechanism is surprisingly not related to blockade of platelet aggregation but rather to triggering of the same signal transduction pathway seen in pre- and postconditioning. We wanted to determine whether this same cardioprotection could be documented in a primate model and whether the protection was limited to P2Y12 receptor antagonists or was a class effect. METHODS: Thirty-one macaque monkeys underwent 90-min LAD occlusion/4-h reperfusion. RESULTS: The platelet P2Y12 receptor blocker cangrelor started just prior to reperfusion significantly decreased infarction by an amount equivalent to that seen with ischemic postconditioning (p < 0.001). For any size of risk zone, infarct size in treated hearts was significantly smaller than that in control hearts. OM2, an investigational murine antibody against the primate collagen receptor glycoprotein (GP) VI, produced similar protection (p < 0.01) suggesting a class effect. Both cangrelor and OM2 were quite effective at blocking platelet aggregation (94 % and 97 %, respectively). CONCLUSIONS: Thus in a primate model in which infarct size could be determined directly platelet anti-aggregatory agents are cardioprotective. The important implication of these investigations is that patients with acute myocardial infarction who are treated with platelet anti-aggregatory agents prior to revascularization may already be in a postconditioned state. This hypothesis may explain why in recent clinical trials postconditioning-mimetic interventions which were so protective in animal models had at best only a modest effect.

Uptake of oxidized low density lipoprotein by CD36 occurs by an actin-dependent pathway distinct from macropinocytosis.

The class B scavenger receptor CD36 has numerous ligands that include modified forms of low density lipoprotein, fibrillar amyloid, apoptotic cells, and Plasmodium falciparum-infected red blood cells, linking this molecule to atherosclerosis, Alzheimer disease, malaria, and other diseases. We studied the signaling events that follow receptor engagement and lead to CD36 and ligand internalization. We show that oxidized low density lipoprotein or antibody-induced clustering of CD36 triggers macropinocytosis and internalization of the receptor-ligand complex. Remarkably, however, CD36 internalization is independent of macropinocytosis and occurs by a novel endocytic mechanism that depends on actin, but not dynamin. This actin-driven endocytosis requires the activation Src family kinases, JNK, and Rho family GTPases, but, unlike macropinocytosis, it is not affected by inhibitors of phosphatidylinositol 3-kinase or Na/H exchange. Manipulation of this unique mode of internalization may prove helpful in the prevention and management of the wide range of diseases in which CD36 is implicated.

Attenuation of infarction in cynomolgus monkeys: preconditioning and postconditioning.

Ischemic pre- (IPC) and post- (IPOC) conditioning are very protective in laboratory animals, but it has not been possible to measure their anti-infarct potency in human hearts. Non-human primates are genetically closer to humans than other laboratory animals, but until now there have been no studies of IPC or IPOC in any primate species. Accordingly the left anterior descending coronary artery of cynomolgus monkeys was occluded for 90 min and reperfused for 4 h. In control animals, only 44% of the risk zone infarcted indicating cynomolgus myocardium is much more resistant to infarction than that of rabbits or rats. The regression line for the infarct-risk zone plot was very linear (r = 0.99), and intersected the risk zone axis at 0.82 cm3. Even small changes in infarct size could be detected as a shift in this line. Collateral flow in 12 monkeys was 6.6% of flow to normal myocardium and not a covariate of infarct size. IPC with two cycles of 10-min coronary occlusion/10-min reperfusion reduced infarction to near zero indicating that the innate resistance to infarction was not caused by constitutive preconditioning. Wortmannin, an antagonist of phosphatidylinositol 3-kinase (PI3-K), administered just before release of the 90-min coronary occlusion attenuated IPC's infarct-sparing effect by approximately 50% suggesting that PI3-K was involved in preconditioning's protection. IPOC with six cycles of 30-s reperfusion/30-s coronary reocclusion, a very protective protocol in most species, was much less protective than IPC. We conclude that ischemic preconditioning is extremely protective in cynomolgus hearts despite their sparse collateralization but, surprisingly, the protocol of IPOC used in this study offers less protection.

Cardiac and skeletal muscle fatty acid transport and transporters and triacylglycerol and fatty acid oxidation in lean and Zucker diabetic fatty rats.

We examined fatty acid transporters, transport, and metabolism in hearts and red and white muscles of lean and insulin-resistant (week 6) and type 2 diabetic (week 24) Zucker diabetic fatty (ZDF) rats. Cardiac fatty acid transport was similar in lean and ZDF hearts at week 6 but was reduced at week 24 (-40%) in lean but not ZDF hearts. Red muscle of ZDF rats exhibited an early susceptibility to upregulation (+66%) of fatty acid transport at week 6 that was increased by 50% in lean and ZDF rats at week 24 but remained 44% greater in red muscle of ZDF rats. In white muscle, no differences were observed in fatty acid transport between groups or from week 6 to week 24. In all tissues (heart and red and white muscle), FAT/CD36 protein and plasmalemmal content paralleled the changes in fatty acid transport. Triacylglycerol content in red and white muscles, but not heart, in lean and ZDF rats correlated with fatty acid transport (r = 0.91) and sarcolemmal FAT/CD36 (r = 0.98). Red and white muscle fatty acid oxidation by isolated mitochondria was not impaired in ZDF rats but was reduced by 18-24% in red muscle of lean rats at week 24. Thus, in red, but not white, muscle of insulin-resistant and type 2 diabetic animals, a marked upregulation in fatty acid transport and intramuscular triacylglycerol was associated with increased levels of FAT/CD36 expression and plasmalemmal content. In heart, greater rates of fatty acid transport and FAT/CD36 in ZDF rats (week 24) were attributable to the inhibition of age-related reductions in these parameters. However, intramuscular triacylglycerol did not accumulate in hearts of ZDF rats. Thus insulin resistance and type 2 diabetes are accompanied by tissue-specific differences in FAT/CD36 and fatty acid transport and metabolism. Upregulation of fatty acid transport increased red muscle, but not cardiac, triacylglycerol accumulation. White muscle lipid metabolism dysregulation was not observed.

Rosiglitazone increases fatty acid oxidation and fatty acid translocase (FAT/CD36) but not carnitine palmitoyltransferase I in rat muscle mitochondria.

Peroxisome proliferator-activated receptors (PPARs) alter the expression of genes involved in regulating lipid metabolism. Rosiglitazone, a PPARgamma agonist, induces tissue-specific effects on lipid metabolism; however, its mode of action in skeletal muscle remains unclear. Since fatty acid translocase (FAT/CD36) was recently identified as a possible regulator of skeletal muscle fatty acid transport and mitochondrial fatty acid oxidation, we examined in this tissue the effects of rosiglitazone infusion (7 days, 1 mg day(-1)) on FAT/CD36 mRNA and protein, its plasmalemmal content and fatty acid transport. In addition, in isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria we examined rates of fatty acid oxidation, FAT/CD36 and carnitine palmitoyltransferase I (CPTI) protein, and CPTI and beta-hydroxyacyl CoA dehydrogenase (beta-HAD) activities. Rosiglitazone did not alter FAT/CD36 mRNA or protein expression, FAT/CD36 plasmalemmal content, or the rate of fatty acid transport into muscle (P > 0.05). In contrast, rosiglitazone increased the rates of fatty acid oxidation in both SS (+21%) and IMF mitochondria (+36%). This was accompanied by concomitant increases in FAT/CD36 in subsarcolemmal (SS) (+43%) and intermyofibrillar (IMF) mitochondria (+46%), while SS and IMF CPTI protein content, and CPTI submaximal and maximal activities (P > 0.05) were not altered. Similarly, citrate synthase (CS) and beta-HAD activities were also not altered by rosiglitazone in SS and IMF mitochondria (P > 0.05). These studies provide another example whereby changes in mitochondrial fatty oxidation are associated with concomitant changes in mitochondrial FAT/CD36 independent of any changes in CPTI. Moreover, these studies identify for the first time a mechanism by which rosiglitazone stimulates fatty acid oxidation in skeletal muscle, namely the chronic, subcellular relocation of FAT/CD36 to mitochondria.

The Fab fragment of a novel anti-GPVI monoclonal antibody, OM4, reduces in vivo thrombosis without bleeding risk in rats.

BACKGROUND: Inhibition of GPVI has been proposed as a useful antithrombotic strategy; however, in vivo proof-of-concept animal studies targeting GPVI are lacking. We evaluated a novel anti-human GPVI monoclonal antibody OM4 Fab in rats. METHODS AND RESULTS: OM4 Fab specifically inhibited collagen-induced aggregation of rat platelets in vitro with an IC50 of 20 to 30 microg/mL but not ADP and AA-induced platelet aggregation. After intravenous administration of OM4 Fab, a rapid inhibition of ex vivo platelet aggregation was observed with a gradual recovery within 60 to 90 minutes which corresponded to the decline in OM4 Fab plasma concentration and time-dependent decrease in platelet-bound OM4 Fab. In contrast to previous reports in mice, intravenous OM4 Fab did not deplete platelet GPVI. Injection of OM4 IgG caused acute thrombocytopenia. In a modified Folts model of cyclic flow reduction in rat carotid artery, the number of complete occlusions was significantly reduced by intravenous administration of OM4 Fab (20 mg/kg) before or after mechanical injury to the vessel, without prolongation of bleeding time. CONCLUSION: Fab fragment of the monoclonal antibody OM4 effectively inhibits collagen induced platelet aggregation in vitro and ex vivo, and in vivo thrombosis in rats without prolonging bleeding time. Antibodies against GPVI may have therapeutic potential, inhibiting thrombosis without prolonging bleeding time.

Highly potent anti-human GPVI monoclonal antibodies derived from GPVI knockout mouse immunization.

Recent progress in the understanding of thrombus formation has suggested an important role for glycoprotein (GP) VI in this process. To clarify the exact role in detail, it is necessary to use specific, high affinity inhibitory antibodies. However, possibly due to the conserved structure of GPVI among species, it has been difficult to obtain potent antibodies. In this study, we developed highly potent anti-human GPVI monoclonal antibodies using GPVI knockout mice for immunization. Fab fragments of these antibodies, named OM1 and OM2, potently inhibit collagen-induced platelet aggregation. The IC(50) values for OM1 and OM2 are 0.6+/-0.05 and 1.7+/-0.5 microg/mL, respectively, showing potency greater than, or equal to that of abciximab (1.7+/-0.3 microg/mL), an anti-GPIIb/IIIa antibody. Fab fragments of OM1 and OM2 also potently inhibit collagen-induced ATP release, thromboxane A(2) formation, and platelet adhesion to immobilized collagen under static and flow conditions. Interestingly, platelet aggregation induced with collagen-related peptide was potently inhibited by OM2 but not OM1, indicating that OM1 recognizes an epitope that is different from collagen-related peptide-binding site on GPVI. These results suggest that OM1 and OM2 may be useful tools to understand the role of GPVI in thrombus formation. Furthermore, these antibodies have the potential to be developed as a new class of therapeutic tool.

Hypoxia-induced fatty acid transporter translocation increases fatty acid transport and contributes to lipid accumulation in the heart.

Protein-mediated LCFA transport across plasma membranes is highly regulated by the fatty acid transporters FAT/CD36 and FABPpm. Physiologic stimuli (insulin stimulation, AMP kinase activation) induce the translocation of one or both transporters to the plasma membrane and increase the rate of LCFA transport. In the hypoxic/ischemic heart, intramyocardial lipid accumulation has been attributed to a reduced rate of fatty acid oxidation. However, since acute hypoxia (15 min) activates AMPK, we examined whether an increased accumulation of intramyocardial lipid during hypoxia was also attributable to an increased rate of LCFA uptake as a result AMPK-induced translocation of FAT/CD36 and FABPpm. In cardiac myocytes, hypoxia (15 min) induced the redistribution of FAT/CD36 from an intracellular pool (LDM) (-25%, P<0.05) to the plasma membranes (PM) (+54%, P<0.05). Hypoxia also induced an increase in FABPpm at the PM (+56%, P<0.05) and a concomitant FABPpm reduction in the LDM (-24%, P<0.05). Similarly, in intact, Langendorff perfused hearts, hypoxia induced the translocation of a both FAT/CD36 and FABPpm to the PM (+66% and +61%, respectively, P<0.05), with a concomitant decline in FAT/CD36 and FABPpm in the LDM (-24% and -23%, respectively, P<0.05). Importantly, the increased plasmalemmal content of these transporters was associated with increases in the initial rates of palmitate uptake into cardiac myocytes (+40%, P<0.05). Acute hypoxia also redirected palmitate into intracellular lipid pools, mainly to PL and TG (+48% and +28%, respectively, P<0.05), while fatty acid oxidation was reduced (-35%, P<0.05). Thus, our data indicate that the increased intracellular lipid accumulation in hypoxic hearts is attributable to both: (a) a reduced rate of fatty acid oxidation and (b) an increased rate of fatty acid transport into the heart, the latter being attributable to a hypoxia-induced translocation of fatty acid transporters.

Ex vivo evaluation of anti-GPVI antibody in cynomolgus monkeys: dissociation between anti-platelet aggregatory effect and bleeding time.

Recent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (> 80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0.2 mg/kg with a slight prolongation of bleeding time (1.3 times baseline value). Furthermore, at 18.8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1.9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5.0 times at 0.35 mg/kg, the lowest effective dose on platelet aggregation. In a pharmacodynamic study, a bolus injection of OM2 at 0.4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exert a potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.

GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism.

Platelet glycoprotein VI (GPVI) is now considered to be a major player in platelet-collagen adhesive interactions leading to thrombus formation. GPVI blockade, or its depletion, has been shown in mice to result in complete protection against arterial thrombosis, without significant prolongation of bleeding time. GPVI may therefore represent a useful antithrombotic target. In order to reaffirm the role of GPVI in platelet-collagen interactions, we developed GPVI(null) mice by targeted disruption methodology. GPVI(null) mice platelets failed to respond to a high dose of fibrillar collagen, or convulxin, a GPVI agonist, but showed a normal response to other agonists such as ADP, PMA and arachidonic acid. We report, for the first time, that a proportion of GPVI(null) mice is protected against lethal thromboembolism, induced by the infusion of a mixture of collagen and epinephrine. Greater than 55% of GPVI(null) mice survived the challenge, whereas the maximal survival from the other genotypes was 17% (n=18 per genotype). Washed platelets obtained from GPVI(null) mice showed >90% reduction in adhesion to fibrillar collagen under static conditions. Platelet adhesion to collagen under dynamic conditions using a high shear rate (2600 s(-1)) was dramatically reduced using blood from GPVI(null) mice, while platelets from wild-type and heterozygous animals showed a similar amount of adhesion. Animals from each genotype had essentially similar tail bleeding time, suggesting that a complete deficiency of GPVI, at least in mice, does not result in an enhanced bleeding tendency. These observations clearly establish that blockade of GPVI may attenuate platelet-collagen interactions without adversely affecting the bleeding time.

Insulin stimulates long-chain fatty acid utilization by rat cardiac myocytes through cellular redistribution of FAT/CD36.

The existence of an intracellular pool of fatty acid translocase (FAT/CD36), an 88-kDa membrane transporter for long-chain fatty acids (FAs), and the ability of insulin to induce translocation events prompted us to investigate the direct effects of insulin on cellular uptake of FA by the heart. Insulin (0.1 nmol/l and higher) increased FA uptake by isolated rat cardiac myocytes by 1.5-fold. This insulin-induced increase in FA uptake was completely blocked by phloretin, sulfo-N-succinimidylpalmitate (SSP), and wortmannin, indicating the involvement of FAT/CD36 and the dependence on phosphatidylinositol-3 (PI-3) kinase activation. Subcellular fractionation of insulin-stimulated cardiac myocytes demonstrated a 1.5-fold increase in sarcolemmal FAT/CD36 and a 62% decrease in intracellular FAT/CD36 with parallel changes in subcellular distribution of GLUT4. Induction of cellular contractions upon electrostimulation at 4 Hz enhanced cellular FA uptake 1.6-fold, independent of PI-3 kinase. The addition of insulin to 4 Hz-stimulated cells further stimulated FA uptake to 2.3-fold, indicating that there are at least two functionally independent intracellular FAT/CD36 pools, one recruited by insulin and the other mobilized by contractions. In conclusion, we have demonstrated a novel role of insulin in cardiac FA utilization. Malfunctioning of insulin-induced FAT/CD36 translocation may be involved in the development of type 2 diabetic cardiomyopathies.

The subcellular compartmentation of fatty acid transporters is regulated differently by insulin and by AICAR.

Cellular fatty acid uptake is facilitated by a number of fatty acid transporters, FAT/CD36, FABPpm and FATP1. It had been presumed that FABPpm, was confined to the plasma membrane and was not regulated. Here, we demonstrate for the first time that FABPpm and FATP1 are also present in intracellular depots in cardiac myocytes. While we confirmed previous work that insulin and AICAR each induced the translocation of FAT/CD36 from an intracellular depot to the PM, only AICAR, but not insulin, induced the translocation of FABPpm. Moreover, neither insulin nor AICAR induced the translocation of FATP1. Importantly, the increased plasmalemmal content of these LCFA transporters was associated with a concomitant increase in the initial rate of palmitate uptake into cardiac myocytes. Specifically, the insulin-stimulated increase in the rate of palmitate uptake (+60%) paralleled the insulin-stimulated increase in plasmalemmal FAT/CD36 (+34%). Similarly, the greater AICAR-stimulated increase in the rate of palmitate uptake (+90%) paralleled the AICAR-induced increase in both plasmalemmal proteins (FAT/CD36 (+40%)+FABPpm (+36%)). Inhibition of palmitate uptake with the specific FAT/CD36 inhibitor SSO indicated that FABPpm interacts with FAT/CD36 at the plasma membrane to facilitate the uptake of palmitate. In conclusion, (1) there appears to be tissue-specific sensitivity to insulin-induced FATP1 translocation, as it has been shown elsewhere that insulin induces FATP1 translocation in 3T3-L1 adipocytes, and (2) clearly, the subcellular distribution of FABPpm, as well as FAT/CD36, is acutely regulated in cardiac myocytes, although FABPpm and FAT/CD36 do not necessarily respond identically to the same stimuli.

Triacylglycerol accumulation in human obesity and type 2 diabetes is associated with increased rates of skeletal muscle fatty acid transport and increased sarcolemmal FAT/CD36.

We examined whether, in human obesity and type 2 diabetes, long chain fatty acid (LCFA) transport into skeletal muscle is upregulated and contributes to an excess intramuscular triacylglycerol accumulation. In giant sarcolemmal vesicles prepared from human skeletal muscle, LCFA transport rates were upregulated approximately 4-fold and were associated with an increased intramuscular triacylglycerol content in obese individuals and in type 2 diabetics. In these individuals, the increased sarcolemmal LCFA transport rate was not associated with an altered expression of FAT/CD36 or FABPpm. Instead, the increase in the LCFA transport rate was associated with an increase in sarcolemmal FAT/CD36 but not sarcolemmal FABPpm. Rates of fatty acid esterification were increased threefold in isolated human muscle strips obtained from obese subjects, while concomitantly rates of fatty acid oxidation were not altered. Thus, the increased rate of fatty acid transport may contribute to the increased rates of triacylglycerol accumulation in human skeletal muscle. The altered FAT/CD36 trafficking in muscle from obese subjects and type 2 diabetics juxtaposes the known alterations in GLUT4 trafficking, i.e., GLUT4 is known to be retained in its intracellular depots while FAT/CD36 is retained at the sarcolemma. This redistribution of FAT/CD36 to the sarcolemma may contribute to the etiology of insulin resistance in human muscle, and hence, FAT/CD36 provides another potential therapeutic target for the prevention and/or treatment of insulin resistance.

Genetic ablation of caveolin-1 confers protection against atherosclerosis.

OBJECTIVE: The development of atherosclerosis is a process characterized by the accumulation of lipids in the form of modified lipoproteins in the subendothelial space. This initiating step is followed by the subsequent recruitment and proliferation of other cell types, including monocytes/macrophages and smooth muscle cells. Here, we evaluate the potential role of caveolae membrane domains in the pathogenesis of atherosclerosis by using apolipoprotein E-deficient (ApoE-/-) mice as a model system. METHODS AND RESULTS: Caveolin-1 (Cav-1) is a principal structural protein component of caveolae membrane domains. To directly assess the in vivo role of caveolae and Cav-1 in atherosclerosis, we interbred Cav-1-/- mice with ApoE-/- mice. Interestingly, loss of Cav-1 resulted in a dramatic >2-fold increase in non-HDL plasma cholesterol levels in the ApoE-/- background. However, despite this hypercholesterolemia, we found that loss of Cav-1 gene expression was clearly protective against the development of aortic atheromas, with up to an approximately 70% reduction in atherosclerotic lesion area. Mechanistically, we demonstrated that loss of Cav-1 resulted in the dramatic downregulation of certain proatherogenic molecules, namely, CD36 and vascular cell adhesion molecule-1. CONCLUSIONS: Taken together, our results indicate that loss of Cav-1 can counteract the detrimental effects of atherogenic lipoproteins. Thus, Cav-1 is a novel target for drug development in the pharmacologic prevention of atheroma formation. Our current data also provide the first molecular genetic evidence to support the hypothesis that caveolar transcytosis of modified lipoproteins (from the blood to the sub-endothelial space) is a critical initiating step in atherosclerosis.

Brain-derived neurotrophic factor is stored in human platelets and released by agonist stimulation.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays critical roles in the survival, growth, and maintenance of brain and peripheral neurons. We report the presence of BDNF protein in human platelets and its release upon agonist stimulation. The BDNF content of washed platelets varied widely, from 3.5 to 67 ng/4 x 10(8) platelets, averaging 25.2 +/- 21.2 ng/4 x 10(8) platelets (mean+/-SD). The BDNF concentration in platelet-poor plasma was low (1.7+/-1.7 ng/ml, n = 11). Thrombin, collagen, the Ca++ ionophore A23187, and shear stress each induced a rapid release of BDNF from platelets. Up to only half of platelet BDNF was secreted upon agonist stimulation, suggesting that platelets may have a non-releasable pool of BDNF, or that the released BDNF binds to a recognition site on the platelet surface and is internalized, as occurs with serotonin. However, the cognate BDNF receptor, TrkB, was not detected in platelets. Nevertheless, the ability of BDNF to bind washed platelets was shown by FACS analysis confocal microscopy and by the binding and apparent internalization of [125I]-BDNF by platelets. A very high affinity site (Kd = 130 x 10(-15) M, approximately 80 sites/platelet) and a moderately high affinity site (Kd = 20 nM, approximately 3750 sites/platelet) were identified. The BDNF content in two megakaryocytic cell lines, DAMI and Meg-01, was only 0.1% of the content measured in platelets. No BDNF mRNA was detected by Northern blotting in these cell lines or in platelets. The pituitary gland was also ruled out as a source for platelet BDNF, since the BDNF content of rat platelets did not decrease 2 weeks after hypophysectomy. Thus, platelet BDNF is not acquired from the megakaryocyte or pituitary gland, but is probably acquired from other sources via the blood circulation. Platelets appear to bind, store and release BDNF upon activation at the site of traumatic injury to facilitate the repair of peripheral nerves or other tissues that contain TrkB.

Expression of glycoprotein VI in vascular endothelial cells.

Glycoprotein (GP) VI, a collagen receptor, plays an important role in collagen-mediated platelet aggregation and adhesion. To date, GPVI expression has been found only in platelets and megakaryocytes. In the present studies, we have demonstrated that GPVI was also expressed in cultured human umbilical vein endothelial cells (HUVEC) at both transcript and protein levels. Using a GPVI-specific probe, a ˜6-kb band was detected in HUVEC as well as in platelets and megakaryoblastic cell lines by Northern blotting. Using polyclonal antibodies raised against platelet GPVI peptides, the same size band (57 kDa) was labeled with convulxin (CVX) after immuno-precipitation in both HUVEC and platelet lysates. In addition, a ˜70-kDa band was also labeled in HUVEC. Surface expression of GPVI in HUVEC was confirmed by flow cytometry with GPVI-specific IgG or by direct labeling with FITC-conjugated CVX. Since HUVEC lack FcRγ chain that forms complex with GPVI in platelets for signaling process, the function of GPVI in vascular endothelial cells remains to be determined.

Platelet P2Y12 blockers confer direct postconditioning-like protection in reperfused rabbit hearts.

BACKGROUND: Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery. METHODS AND RESULTS: The P2Y12 (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel's protection. Neither aspirin nor heparin was protective. CONCLUSIONS: Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC's protective signaling to prevent reperfusion injury. If true, patients receiving P2Y12 inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs.

Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

BACKGROUND: Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation. METHODOLOGY/PRINCIPAL FINDINGS: We utilized the Mx-cre;Cdc42(lox/lox) inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42(+/+) mice. Platelets isolated from Cdc42(-/-), as compared to Cdc42(+/+), mice exhibited (a) diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b) inhibition of filopodia formation on immobilized CRP or fibrinogen, (c) inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d) inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e) minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42(-/-) mice compared with Cdc42(+/+) mice. CONCLUSION/SIGNIFICANCE: Our data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

FAT/CD36 expression is not ablated in spontaneously hypertensive rats.

There is doubt whether spontaneously hypertensive rats (SHR; North American strain) are null for fatty acid translocase (FAT/CD36). Therefore, we examined whether FAT/CD36 is expressed in heart, muscle, liver and adipose tissue in SHR. Insulin resistance was present in SHR skeletal muscle. We confirmed that SHR expressed aberrant FAT mRNAs in key metabolic tissues; namely, the major 2.9 kb transcript was not expressed, but 3.8 and 5.4 kb transcripts were present. Despite this, FAT/CD36 protein was expressed in all tissues, although there were tissue-specific reductions in FAT/CD36 protein expression and plasmalemmal content, ranging from 26-85%. Fatty acid transport was reduced in adipose tissue (-50%) and was increased in liver (+47%). Normal rates of fatty acid transport occurred in heart and muscle, possibly due to compensatory upregulation of plasmalemmal fatty acid binding protein (FABPpm) in red (+123%) and white muscle (+110%). In conclusion, SHRs (North American strain) are not a natural FAT/CD36 null model, the North American strain of SHR express FAT/CD36, albeit at reduced levels.