Imala Alwis

Bcl-xL–inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets

BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)β(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.

Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin αIIbβ3 in Mice

Background: Dok proteins are negative regulators of immunoreceptor signaling and, potentially, integrin adhesion receptors. Results: Deficiency of Dok-2 results in enhanced shear-dependent integrin adhesion in platelets, leading to accelerated platelet thrombus growth. Conclusion: Dok-2 is a shear-specific negative regulator of blood clot formation. Significance: Dok-2 regulates biomechanical platelet adhesion, and targeting this molecule may provide new avenues to regulate thrombosis. The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbβ3, raising the possibility that it participates in integrin αIIbβ3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbβ3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbβ3 affinity regulation was unaltered in Dok-2−/− platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbβ3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.

Neutrophil macroaggregates promote widespread pulmonary thrombosis after gut ischemia

Neutrophil macroaggregates induce a distinct arterial-venous thrombotic response in the lung after gut ischemia. Rip n’ roll Ischemia in critically ill patients can result in thrombosis of unrelated organs, which is partially due to neutrophil recruitment. Yuan et al. combined intravital microscopy of thrombosis after gut ischemia-reperfusion injury with samples from acute respiratory distress syndrome patients. They observed that rolling neutrophils grab and rip fragments from phosphatidylserine-expressing dying platelets, which leads to macroaggregates. These macroaggregates, in turn, induce thrombosis and were not able to be targeted by conventional therapies such as aspirin. Conversely, targeting the necrotic factor cyclophilin D did have beneficial effects. These studies reveal new thrombotic biology and suggest the development of alternatively targeted therapies to prevent distant organ injury. Gut ischemia is common in critically ill patients, promoting thrombosis and inflammation in distant organs. The mechanisms linking hemodynamic changes in the gut to remote organ thrombosis remain ill-defined. We demonstrate that gut ischemia in the mouse induces a distinct pulmonary thrombotic disorder triggered by neutrophil macroaggregates. These neutrophil aggregates lead to widespread occlusion of pulmonary arteries, veins, and the microvasculature. A similar pulmonary neutrophil-rich thrombotic response occurred in humans with the acute respiratory distress syndrome. Intravital microscopy during gut ischemia-reperfusion injury revealed that rolling neutrophils extract large membrane fragments from remnant dying platelets in multiple organs. These platelet fragments bridge adjacent neutrophils to facilitate macroaggregation. Platelet-specific deletion of cyclophilin D, a mitochondrial regulator of cell necrosis, prevented neutrophil macroaggregation and pulmonary thrombosis. Our studies demonstrate the existence of a distinct pulmonary thrombotic disorder triggered by dying platelets and neutrophil macroaggregates. Therapeutic targeting of platelet death pathways may reduce pulmonary thrombosis in critically ill patients.

Valproic acid selectively increases vascular endothelial tissue-type plasminogen activator production and reduces thrombus formation in the mouse

Essentials Stimulating endogenous fibrinolysis could be a novel antithrombotic strategy. The effect of valproic acid on endothelial tissue plasminogen activator in mice was investigated. Valproic acid increased tissue plasminogen activator expression in vascular endothelium. Valproic acid reduced fibrin deposition and thrombus formation after vascular injury.

Thromboinflammatory Functions of Platelets in Ischemia–Reperfusion Injury and Its Dysregulation in Diabetes

&NA; Ischemia‐reperfusion (IR) injury is a common complication of a variety of cardiovascular diseases, including ischemic stroke and myocardial infarction (MI). While timely re‐establishment of blood flow in a thrombosed artery is the primary goal of acute therapy in these diseases, paradoxically, reperfusion of ischemic tissue can cause widespread microvascular dysfunction that significantly exacerbates organ damage. Reperfusion injury is associated with activation of the humoral and cellular components of the hemostatic and innate immune systems and also with excessive reactive oxygen species production, endothelial dysfunction, thrombosis, and inflammation. Platelets are critical mediators of thromboinflammation during reperfusion injury and a hyperactive platelet phenotype may contribute to an exaggerated IR injury response. This is particularly relevant to diabetes which is characteristically associated with hyperactive platelets, significantly worse IR injury, increased organ damage, and increased risk of death. However, the mechanisms underlying vulnerability to IR injury in diabetic individuals is not well defined, nor the role of “diabetic platelets” in this process. This review summarizes recent progress in understanding the role of platelets in promoting microvascular dysfunction and inflammation in the context of IR injury. Furthermore, the authors discuss aspects of the thromboinflammatory function of platelets that are dysregulated in diabetes. They conclude that diabetes likely enhances the capacity of platelets to mediate microvascular thrombosis and inflammation during IR injury, which has potentially important implications for the future design of antiplatelet agents that can reduce microvascular dysfunction and inflammation.

Compression force sensing regulates integrin αiIbβ3 adhesive function on diabetic platelets

Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin αIIbβ3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to αIIbβ3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.Diabetes is associated with an increased thrombotic response, but the mechanism is unknown. Here the authors demonstrate that compressive force activates integrin αIIbβ3 on discoid diabetic platelets and that platelet aggregates can be eliminated by PI 3-kinase inhibition, but not by anti-thrombotics aspirin or clopidogrel.

Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion

The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.

Endogenous fibrinolysis facilitates clot retraction in vivo

Clot retraction refers to the process whereby activated platelets transduce contractile forces onto the fibrin network of a thrombus, which over time increases clot density and decreases clot size. This process is considered important for promoting clot stability and maintaining blood vessel patency. Insights into the mechanisms regulating clot retraction at sites of vascular injury have been hampered by a paucity of in vivo experimental models. By pairing localized vascular injury with thrombin microinjection in the mesenteric circulation of mice, we have demonstrated that the fibrin network of thrombi progressively compacts over a 2-hour period. This was a genuine retraction process, as treating thrombi with blebbistatin to inhibit myosin IIa-mediated platelet contractility prevented shrinkage of the fibrin network. Real-time confocal analysis of fibrinolysis after recombinant tissue-type plasminogen activator (tPA) administration revealed that incomplete proteolysis of fibrin polymers markedly facilitated clot retraction. Similarly, inhibiting endogenous fibrinolysis with tranexamic acid reduced retraction of fibrin polymers in vivo. In vitro clot retraction experiments indicated that subthreshold doses of tPA facilitated clot retraction through a plasmin-dependent mechanism. These effects correlated with changes in the elastic modulus of fibrin clots. These findings define the endogenous fibrinolytic system as an important regulator of clot retraction, and show that promoting clot retraction is a novel and complementary means by which fibrinolytic enzymes can reduce thrombus size.

Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation–desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.