Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease

MYH9-related disease patients with mutations in the contractile protein non-muscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild to moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in MYH9-related disease patients, and treatment with tranexamic acid can improve the hemostatic function.

A novel role for endoplasmic reticulum protein 46 (ERp46) in platelet function and arterial thrombosis in mice

Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has three CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, ATP release, and αIIbβ3 activation. ERp46 protein potentiated αIIbβ3 activation, platelet aggregation and ATP release, while inactive ERp46 inhibited these processes. ERp46-knockout mice had prolonged tail-bleeding times, and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbβ3 activation, platelet aggregation, ATP release and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the three active sites. Platelet aggregation stimulated by an αIIbβ3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbβ3 by surface plasmon resonance but poorly to platelets lacking αIIbβ3, and physically associated with αIIbβ3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the β3 subunit than other PDIs, and in contrast to PDI generated thiols in β3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in β3 implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in β3 implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbβ3 activation by targeting disulfide bonds.

Platelet binding to polymerizing fibrin is avidity driven and requires activated αIIbβ3 but not fibrin cross-linking

The molecular basis of platelet-fibrin interactions remains poorly understood despite the predominance of fibrin in thrombi. We have studied the interaction of platelets with polymerizing fibrin by adding thrombin to washed platelets in the presence of the peptide RGDW, which inhibits the initial platelet aggregation mediated by fibrinogen binding to αIIbβ3 but leaves intact a delayed increase in light transmission (delayed wave; DW) as platelets interact with the polymerizing fibrin. The DW was absent in platelets from a patient with Glanzmann thrombasthenia, indicating a requirement for αIIbβ3. The DW required αIIbb3 activation and it was inhibited by the αIIbβ3 antagonists eptifibatide and the monoclonal antibody (mAb) 7E3, but only at much higher concentrations than needed to inhibit platelet aggregation initiated by a thrombin receptor activating peptide (T6). Surface plasmon resonance and scanning electron microscopy studies both supported fibrin having greater avidity for αIIbβ3 than fibrinogen rather than greater affinity, consistent with fibrin’s multivalency. mAb 10E5, a potent inhibitor of T6-induced platelet aggregation, did not inhibit the DW, suggesting that fibrin differs from fibrinogen in its mechanism of binding. Inhibition of factor XIII–mediated fibrin cross-linking by >95% reduced the DW by only 32%. Clot retraction showed a pattern of inhibition similar to that of the DW. We conclude that activated αIIbβ3 is the primary mediator of platelet-fibrin interactions leading to clot retraction, and that the interaction is avidity driven, does not require fibrin cross-linking, and is mediated by a mechanism that differs subtly from that of the interaction of αIIbβ3 with fibrinogen.

Force-Independent Cleavage of Talin by Calpain Promotes Platelet-Mediated Fibrin Clot Contraction

Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino-termini and mass spectrometry to identify proteolytically-cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, fourteen were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a "mechanosensitive clutch" connecting integrins bound to the extracellular matrix to the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of four- and five-helix bundles. The bundles contain 11 vinculin binding sites (VBS), each of which is an α-helix packed into a bundle interior and requiring a structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Thus, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.

The binding of autotaxin to integrins mediates hyperhomocysteinemia-potentiated platelet activation and thrombosis

Hyperhomocysteinemia (HHcy) is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Here, a human medical examination report showed that elevated human plasma Hcy levels were positively correlated with enhanced blood coagulation and platelet activity, suggesting that humans with HHcy are more prone to thrombus formation at the sites of vascular injury. Accordingly, we observed accelerated platelet activation, primary hemostasis, and thrombus formation both in acute and chronic HHcy ApoE-/- mice. Upon Hcy administration in C57BL/6J mice, platelet aggregation, spreading, and clot retraction were markedly promoted. More importantly, homocysteine (Hcy) increased the affinity of platelet integrin αIIbβ3 with ligands and enhanced integrin outside-in signaling by promoting membrane phosphatidylserine (PS) exposure in vitro. Mechanistically, lipidomics analysis showed that lysophosphatidylcholines were the primary metabolites leading to clustering of HHcy-stimulated platelets. Cytosolic phospholipase A2 (cPLA2) activity and autotaxin (ATX, a secreted lysophospholipase D) secretion were upregulated by Hcy, leading to membrane phospholipid hydrolysis and PS exposure. Moreover, secreted ATX directly interacted with integrin β3. Inhibitors of cPLA2 and ATX activity blocked integrin αIIbβ3 outside-in signaling and thrombosis in HHcy ApoE-/- mice. This study identifies a novel mechanism by which HHcy promotes platelet membrane phospholipid catabolism and extracellular ATX secretion to activate integrin outside-in signaling, consequently to exaggerate thrombosis. This study reveals an innovative approach to treat HHcy-related thrombotic diseases.

Clot Retraction: Cellular Mechanisms and Inhibitors, Measuring Methods, and Clinical Implications

Platelets have important functions in hemostasis. Best investigated is the aggregation of platelets for primary hemostasis and their role as the surface for coagulation leading to fibrin- and clot-formation. Importantly, the function of platelets does not end with clot formation. Instead, platelets are responsible for clot retraction through the concerted action of the activated αIIbβ3 receptors on the surface of filopodia and the platelet’s contractile apparatus binding and pulling at the fibrin strands. Meanwhile, the signal transduction events leading to clot retraction have been investigated thoroughly, and several targets to inhibit clot retraction have been demonstrated. Clot retraction is a physiologically important mechanism allowing: (1) the close contact of platelets in primary hemostasis, easing platelet aggregation and intercellular communication, (2) the reduction of wound size, (3) the compaction of red blood cells to a polyhedrocyte infection-barrier, and (4) reperfusion in case of thrombosis. Several methods have been developed to measure clot retraction that have been based on either the measurement of clot volume or platelet forces. Concerning the importance of clot retraction in inborn diseases, the failure of clot retraction in Glanzmann thrombasthenia is characterized by a bleeding phenotype. Concerning acquired diseases, altered clot retraction has been demonstrated in patients with coronary heart disease, stroke, bronchial asthma, uremia, lupus erythematodes, and other diseases. However, more studies on the diagnostic and prognostic value of clot retraction with methods that have to be standardized are necessary.

Naringenin Inhibits Platelet Activation and Arterial Thrombosis Through Inhibition of Phosphoinositide 3-Kinase and Cyclic Nucleotide Signaling

Citrus flavanoids intake can reduce the risk of cardiovascular diseases. Naringenin, a natural predominant flavonoid abundant in citrus fruits, possesses protective effects against atherothrombotic diseases. As platelet activation plays central roles in atherothrombogenesis, we studied the effects of naringenin on platelet activation, signaling, thrombosis and hemostasis. Naringenin dose-dependently inhibited agonist-induced platelet aggregation in vitro, and exhibited more-potent efficacy on ADP-induced platelet aggregation. It also suppressed platelet aggregation stimulated by ADP ex vivo. Naringenin inhibited ADP-induced platelet α-granule secretion, fibrinogen binding, intracellular calcium mobilization and platelet adhesion on collagen-coated surface. Naringenin also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in integrin signaling. Mechanism studies indicated that naringenin suppressed PI3K-mediated signaling and phosphodiesterase activity in platelets, in addition to increasing cGMP levels and VASP phosphorylation at Ser239. Furthermore, naringenin-induced VASP phosphorylation and inhibition of platelet aggregation were reversed by a PKA inhibitor treatment. Interestingly, naringenin inhibited thrombus formation in the (FeCl3)-induced rat carotid arterial thrombus model, but not cause a prolonged bleeding time in mice. This study suggests that naringenin may represent a potential antiplatelet agent targeting PI3K and cyclic nucleotide signaling, with a low bleeding risk.

Thymosin β4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes (MKs) but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin β4 (encoded by Tmsb4x) is one of the two main G-actin sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin β4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. MK numbers in the bone marrow (BM) and spleen were unaltered, however, Tmsb4x KO MKs showed defective proplatelet formation in vitro and in vivo. Thymosin β4 deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein (GP) VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin β4 for actin dynamics during platelet biogenesis, platelet activation downstream of GPVI and thrombus stability.

Roxadustat Does Not Affect Platelet Production, Activation, and Thrombosis Formation

Objective: Roxadustat is a new medication for the treatment of renal anemia. EPO (erythropoietin)—the current treatment standard—has been reported to enhance platelet activation and production. However, to date, the effect of roxadustat on platelets is unclear. To address this deficiency, herein, we have evaluated the effect of roxadustat on platelet production and function. Approach and Results: We performed several mouse platelet functional assays in the presence/absence of in vitro and in vivo roxadustat treatment. Both healthy and 5/6 nephrectomized mice were utilized. The effect of roxadustat on platelet function of healthy volunteers and chronic kidney disease patients was also evaluated. For platelet production, megakaryocyte maturation and proplatelet formation were assayed in vitro. Peripheral platelet and bone marrow megakaryocyte counts were also determined. We found that roxadustat could not stimulate washed platelets directly, and platelet aggregation, spreading, clot retraction, and P-selectin/JON/A exposure were similar with or without in vitro or in vivo roxadustat treatment among both healthy and 5/6 nephrectomized mice. In vivo mouse thrombosis models were additionally performed, and no differences were detected between the vehicle and roxadustat treatment groups. EPO, which was considered a positive control in the present study, promoted platelet function and production as reported previously. Megakaryocyte maturation and proplatelet formation were also not significantly different between control mice and those treated with roxadustat. After receiving roxadustat for 14 days, no difference in the peripheral platelet count was observed in the mice. Conclusions: Administration of roxadustat has no significant impact on platelet production and function.